KR20220119362A - Compositions and methods related to engineered Fc-antigen binding domain constructs targeting CD38 - Google Patents

Abstract

Fc-antigen binding constructs having a CD38 binding domain and two or more Fc domains are described, and methods for using such constructs are likewise described. Also described are the polypeptides that make up such constructs. The Fc domain monomers included in the construct may include amino acid substitutions that promote homodimerization or heterodimerization.

Description

Compositions and methods related to engineered Fc-antigen binding domain constructs targeted to CD38

CD38 is a type II transmembrane glycoprotein that is expressed at high density on normal and malignant plasmablasts and plasma cells and at low levels on certain lymphoid and myeloid cells. Darzalex (daratumumab) is an anti-CD38 cytolytic monoclonal antibody approved for relapsed, refractory multiple myeloma and newly diagnosed multiple myeloma.

The present invention features compositions and methods for combining a CD38 binding domain with at least two Fc domains to create new therapeutic agents with unique biological activity.

In some cases, the present invention provides a known CD38 targeted single Fc-domain containing therapeutic agent, e.g., combining the CD38 binding domain of a known therapeutic CD38 antibody with at least two Fc domains to have greater biological activity than a known CD38 antibody. Consider creating new therapeutics. To generate such constructs, the present invention provides various methods for assembling constructs having at least two, eg, multiple, Fc domains, including homodimerization and heterodimerization of such constructs. Controlled, it provides a variety of methods for assembling molecules of distinct sizes from a limited number of polypeptides. The properties of these constructs allow for the efficient production of substantially homogeneous pharmaceutical compositions. Such homogeneity in the pharmaceutical composition is desirable to ensure the safety, efficacy, uniformity, and reliability of the pharmaceutical composition.

In a first aspect, the invention features an Fc-antigen binding domain construct comprising enhanced effector function, said Fc-antigen binding domain construct comprising a CD38 binding domain and a second Fc domain by a linker wherein the Fc-antigen binding domain construct comprises a first Fc domain linked to an antibody-dependent cytotoxicity (ADCC) assay, antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ) has improved effector function compared to the construct with a single Fc domain and a CD38 binding domain in the assay.

In a second aspect, the invention features a composition comprising a substantially homogeneous population of Fc-antigen binding domain constructs comprising a CD38 binding domain and a first Fc domain linked to a second Fc domain by a linker. .

In a third aspect, the invention features an Fc-antigen binding domain construct comprising a CD38 binding domain and a first Fc domain linked to a second Fc domain by a linker, said Fc-antigen binding domain construct comprising a single Fc domain and biological activity not exhibited by a construct having said CD38 binding domain.

In a fourth aspect, the invention features a composition comprising a substantially homogeneous population of Fc-antigen binding domain constructs, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii ) a second Fc domain monomer, and iii) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a CD38 binding domain linked to said first, second, or third polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain; The second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.

In some embodiments of the fourth aspect, the CD38 binding domain is linked to a first polypeptide and a second polypeptide or a third polypeptide, is linked to a second polypeptide and a third polypeptide, or the CD38 binding domain is a first polypeptide, a second polypeptide , and a third polypeptide.

In a fifth aspect, the invention features an Fc-antigen binding domain construct comprising enhanced effector function, said Fc-antigen binding domain construct comprising: a) i) a first Fc domain monomer, ii) a second Fc a first polypeptide comprising a domain monomer, and iii) a linker connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a CD38 binding domain linked to said first, second, or third polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain; The second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain, wherein the Fc-antigen binding domain construct is tested in an antibody-dependent cytotoxicity (ADCC) assay, antibody-dependent cellular phagocytosis ( ADCP), and/or enhanced effector function compared to constructs with a single Fc domain and a CD38 binding domain in a complement-dependent cytotoxicity (CDC) assay.

In some embodiments of the fifth aspect, the single Fc domain construct is an antibody.

In a sixth aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a CD38 binding domain linked to said first, second, or third polypeptide;

The first Fc domain monomer and the third Fc domain monomer are combined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are combined to form a second Fc domain, and the Fc - the antigen binding domain construct comprises a single Fc domain and a biological activity not exhibited by the construct with said CD38 binding domain.

In some embodiments of the sixth aspect, the biological activity is an Fc receptor mediated effector function, such as ADCC, ADCP and/or CDC activity (eg, ADCC and ADCP activity, ADCC and CDC activity, ADCP and CDC activity, or ADCC, ADCP, and CDC activity).

In a seventh aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a spacer connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a CD38 binding domain linked to said first, second, or third polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain; The second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.

In some embodiments of the fifth, sixth, and seventh aspects of the invention, the CD38 binding domain is linked to a first polypeptide and a second polypeptide or a third polypeptide, is linked to a second polypeptide and a third polypeptide, or , the CD38 binding domain is linked to the first polypeptide, the second polypeptide, and the third polypeptide.

In some embodiments of the first, second, third, and fourth aspects of the invention, the CD38 binding domain is a Fab or V _H of the Fab.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the binding domain is part of the amino acid sequence of the first, second, or third polypeptide, and in some embodiments, The CD38 binding domain is an scFv.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the CD38 binding domain comprises a V _H domain and a C _H 1 domain, wherein the V _H and C _H 1 domains are It is part of the amino acid sequence of a first, second, or third polypeptide. In some embodiments, the CD38 binding domain further comprises a _VL domain, and in some embodiments the Fc-antigen binding domain construct comprises a fourth polypeptide comprising a _VL domain. In some embodiments, the V _H domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, or the V _H domain comprises a CDR of a V _H domain comprising the sequences of an antibody set forth in Table 2 -H1, CDR-H2, and CDR-H3, or the V _H domain comprises CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody set forth in Table 2, wherein the CDR-H1, CDR the V _H sequence excluding the -H2 and CDR-H3 sequences is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H sequence of the antibody set forth in Table 2, or The V _H domain comprises the V _H sequence of an antibody shown in Table 2.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the CD38 binding domain comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR set forth in Table 1. _-L2 , and a set of CDR-L3 sequences, or the CD38 binding domain comprises a CDR-H1, CDR-H2, CDR-H3, CDR- _L1 , A V _H domain comprising CDR-L2, and a CDR-L3 sequence, or a CD38 binding domain comprising CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody shown in Table 2, and a _VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the V _L sequence of a given antibody, and comprising a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and The V _H and V _L domain sequences, excluding the CDR-L3 sequences, are at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H and V _L sequences of the antibodies set forth in Table 2 Alternatively, the CD38 binding domain comprises the set of V _H and V _L sequences of an antibody shown in Table 2.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the Fc-antigen binding domain construct further comprises an IgG _CL antibody constant domain and an IgG _CH 1 antibody constant domain. wherein the IgG _CH 1 antibody constant domain is attached to the N-terminus of the first polypeptide or the second polypeptide by a linker.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the first Fc domain monomer and the third Fc domain monomer are formed between the first Fc domain monomer and the third Fc domain monomer. Complementary dimerization selectivity module to promote dimerization.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the second Fc domain monomer and the fourth Fc domain monomer are formed between the second Fc domain monomer and the fourth Fc domain monomer. Complementary dimerization selectivity module to promote dimerization.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the dimerization selectivity module comprises an engineered cavity into the C _H 3 domain of one of the Fc domain monomers and comprising an engineered protuberance into the C _H 3 domain of the other of the Fc domain monomers, wherein the engineered cavity and the engineered protuberance form a protuberance-into-cavity pair into the cavity of the Fc domain monomers. positioned to form. In some embodiments, the engineered protrusion comprises at least one modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and wherein the engineered cavity comprises Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and at least one modification selected from T394S. In some embodiments, one of the Fc domain monomers comprises Y407V and Y349C and the other of the Fc domain monomers comprises T366W and S354C.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the dimerization selectivity module comprises a negatively charged amino acid into a C _H 3 domain of one of the domain monomers and an Fc domain. a positively charged amino acid into the C _H 3 domain of the other of the monomers, wherein the negatively and positively charged amino acids are positioned to promote formation of the Fc domain. In some embodiments, each of the first and third Fc domain monomers comprises D399K and either K409D or K409E, or each of the first and third Fc domain monomers comprises K392D and D399K or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370E, or each of the first Fc domain monomer and the third Fc domain monomer comprises D356K and K439D, or each of the first Fc domain the monomer and the third Fc domain monomer comprise K392E and D399K, or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370D, or each of the first Fc domain monomer and the third Fc domain monomer comprises: D356K and K439E, or each of the second and fourth Fc domain monomers comprises S354C and T366W and each of the third and fourth polypeptides comprises Y349C, T366S, L368A, and Y407V, respectively wherein the third and fourth polypeptides comprise S354C and T366W and each of the second Fc domain monomer and the fourth Fc domain monomer comprises Y349C, T366S, L368A, and Y407V, or each of the second Fc domain monomer and the first the 4 Fc domain monomers comprise E357K or E357R and each of the third and fourth polypeptides comprises K370D or K370E, or each of the second and fourth Fc domain monomers comprises K370D or K370E and each the third and fourth polypeptides comprise E357K or 357R, or each of the second and fourth Fc domain monomers comprises K409D or K409E and each of the third and fourth polypeptides comprises D399K or D399R or each of the second Fc domain monomer and the fourth F The c domain monomer comprises D399K or D399R and each of the third and fourth polypeptides comprises K409D or K409E.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the second polypeptide and the third polypeptide have the same amino acid sequence.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the one or more linkers in the Fc-antigen binding domain construct is a bond.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a spacer. 일부 실시 형태에서, 스페이서는 서열 GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGG. In some embodiments, the spacer is a glycine spacer, for example consisting of 4-30, 8-30, or 12-30 glycine residues, such as a spacer consisting of 20 glycine residues.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the CD38 binding domain is linked to the Fc domain monomer by a linker. In some embodiments, the linker is a spacer.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at EU position 1253. In some embodiments, each amino acid modification at position I253 is independently I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y. In some embodiments, each amino acid modification at position 1253 is 1253A.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at EU position R292. In some embodiments, each amino acid modification at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid modification at position R292 is R292P.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, at least one of the Fc domain monomers is an IgG hinge domain, an IgG _CH 2 antibody constant domain, and an IgG _CH 3 antibody constant domains. In some embodiments, each Fc domain monomer comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, the IgG has a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the N-terminal Asp in each of the fourth, fifth, sixth, and seventh polypeptides is mutated to Gln do.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, at least one of the fourth, fifth, sixth, and seventh polypeptides lacks a C-terminal lysine . In some embodiments, each of the fourth, fifth, sixth, and seventh polypeptides lacks a C-terminal lysine.

In some embodiments of the fourth, fifth, sixth, and seventh aspects of the invention, the Fc-antigen binding domain construct is linked to the N-terminus or C-terminus of one or more of the polypeptides by a linker. an albumin-binding peptide.

In an eighth aspect, the invention features a cell culture medium comprising a population of Fc-antigen binding domain constructs, wherein at least 50% of said Fc-antigen binding domain constructs on a molar basis are structurally identical, said The Fc-antigen binding domain construct is present in the culture medium at a concentration of at least 0.1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, or 100 mg/L.

In some embodiments of the eighth aspect of the invention, at least 75%, at least 85%, or at least 95% of the Fc-antigen binding domain constructs on a molar basis are structurally identical.

In a ninth aspect, the invention features a cell culture medium comprising a population of Fc-antigen binding domain constructs, wherein at least 50% of said Fc-antigen binding domain constructs on a molar basis comprise a) i) a first a first polypeptide comprising an Fc domain monomer, ii) a second Fc domain monomer, and iii) a linker connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a CD38 binding domain linked to said first, second, or third polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain; The second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.

In some embodiments of the ninth aspect of the invention, at least 75%, at least 85%, or at least 95% of the Fc-antigen binding domain constructs on a molar basis comprise the first Fc domain, the second Fc domain, and the CD38 binding domain. includes

In a tenth aspect, the invention features a method of making an Fc-antigen binding domain construct, said method comprising: a) (1) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer; (2) a second polypeptide comprising a third Fc domain monomer; (3) a third polypeptide comprising a fourth Fc domain monomer; and (4) culturing a host cell expressing the CD38 binding domain, wherein the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain, and the second Fc domain monomer and the the fourth Fc domain monomers combine to form a second Fc domain; wherein said CD38 binding domain is linked to said first polypeptide, second polypeptide, or third polypeptide to form an Fc-antigen binding domain construct; wherein at least 50% of the Fc-antigen binding domain construct in the cell culture supernatant is structurally identical on a molar basis, and b) purifying the Fc-antigen binding domain construct from the cell culture supernatant; include

In some embodiments of the ninth and tenth aspects of the present invention, the CD38 binding domain is linked to a first polypeptide and a second polypeptide or a third polypeptide, is linked to a second polypeptide and a third polypeptide, or the CD38 binding domain is a first polypeptide, a second polypeptide, and a third polypeptide.

In some embodiments of the ninth and tenth aspects of the invention, the CD38 binding domain is Fab or V _H .

In some embodiments of the ninth and tenth aspects of the invention, the CD38 binding domain is part of the amino acid sequence of the first, second, or third polypeptide, and in some embodiments, the CD38 binding domain is an scFv.

In some embodiments of the ninth and tenth aspects of the invention, the CD38 binding domain comprises a V _H domain and a C _H 1 domain, wherein the V _H and C _H 1 domains are the first, second, or third polypeptides. It is part of the amino acid sequence of In some embodiments, the CD38 binding domain further comprises a _VL domain, and in some embodiments the Fc-antigen binding domain construct comprises a fourth polypeptide comprising a _VL domain. In some embodiments, the V _H domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, or the V _H domain comprises a CDR of a V _H domain comprising the sequences of an antibody set forth in Table 2 -H1, CDR-H2, and CDR-H3, or the V _H domain comprises CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody set forth in Table 2, wherein the CDR-H1, CDR the V _H sequence excluding the -H2 and CDR-H3 sequences is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H sequence of the antibody set forth in Table 2, or The V _H domain comprises the V _H sequence of an antibody shown in Table 2.

In some embodiments of the ninth and tenth aspects of the invention, the CD38 binding domain comprises one of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences shown in Table 1. or the CD38 binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from the set of V _H and V _L sequences of the antibodies shown in Table 2. or the CD38 binding domain comprises a V _H domain comprising the CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of the antibody shown in Table 2, and the CDR- of the _VL sequence of the antibody shown in Table 2 a V _L domain comprising L1, CDR-L2, and CDR-L3, and V _H excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences; the V _L domain sequence is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H and V _L sequences of the antibody set forth in Table 2, or the CD38 binding domain is contains the set of V _H and V _L sequences of the antibody presented in

In some embodiments of the ninth and tenth aspects of the invention, the Fc-antigen binding domain construct further comprises an IgG C _L antibody constant domain and an IgG C _H 1 antibody constant domain, wherein the Fc-antigen binding domain construct further comprises an IgG C _H 1 antibody constant domain. The domain is attached to the N-terminus of the first polypeptide or the second polypeptide by a linker.

In some embodiments of the ninth and tenth aspects of the invention, the first Fc domain monomer and the third Fc domain monomer undergo complementary dimerization to promote dimerization between the first Fc domain monomer and the third Fc domain monomer. Includes a selectivity module.

In some embodiments of the ninth and tenth aspects of the invention, the second Fc domain monomer and the fourth Fc domain monomer undergo complementary dimerization to promote dimerization between the second Fc domain monomer and the fourth Fc domain monomer. Includes a selectivity module.

In some embodiments of the ninth and tenth aspects of the invention, the dimerization selectivity module is an engineered cavity into the C _H 3 domain of one of the Fc domain monomers and the C _H 3 domain of the other of the Fc domain monomers. an engineered protrusion into the cavity, wherein the engineered cavity and the engineered protrusion are positioned to form a pair of protrusions into the cavity of the Fc domain monomers. In some embodiments, the engineered protrusion comprises at least one modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and wherein the engineered cavity comprises Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and at least one modification selected from T394S. In some embodiments, one of the Fc domain monomers comprises Y407V and Y349C and the other of the Fc domain monomers comprises T366W and S354C.

In some embodiments of the ninth and tenth aspects of the invention, the dimerization selectivity module comprises a negatively charged amino acid into the C _H 3 domain of one of the domain monomers and C _H 3 of the other of the Fc domain monomers. positively charged amino acids into the domain, wherein the negatively and positively charged amino acids are positioned to promote formation of the Fc domain. In some embodiments, each of the first and third Fc domain monomers comprises D399K and either K409D or K409E, or each of the first and third Fc domain monomers comprises K392D and D399K or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370E, or each of the first Fc domain monomer and the third Fc domain monomer comprises D356K and K439D, or each of the first Fc domain the monomer and the third Fc domain monomer comprise K392E and D399K, or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370D, or each of the first Fc domain monomer and the third Fc domain monomer comprises: D356K and K439E, or each of the second and fourth Fc domain monomers comprises S354C and T366W and each of the third and fourth polypeptides comprises Y349C, T366S, L368A, and Y407V, respectively wherein the third and fourth polypeptides comprise S354C and T366W and each of the second Fc domain monomer and the fourth Fc domain monomer comprises Y349C, T366S, L368A, and Y407V, or each of the second Fc domain monomer and the first the 4 Fc domain monomers comprise E357K or E357R and each of the third and fourth polypeptides comprises K370D or K370E, or each of the second and fourth Fc domain monomers comprises K370D or K370E and each the third and fourth polypeptides comprise E357K or 357R, or each of the second and fourth Fc domain monomers comprises K409D or K409E and each of the third and fourth polypeptides comprises D399K or D399R or each of the second Fc domain monomer and the fourth F The c domain monomer comprises D399K or D399R and each of the third and fourth polypeptides comprises K409D or K409E.

In some embodiments of the ninth and tenth aspects of the invention, the second polypeptide and the third polypeptide have the same amino acid sequence.

In some embodiments of the ninth and tenth aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a bond.

In some embodiments of the ninth and tenth aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a spacer. 일부 실시 형태에서, 스페이서는 서열 GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGG. In some embodiments, the spacer is a glycine spacer, for example consisting of 4-30, 8-30, or 12-30 glycine residues, such as a spacer consisting of 20 glycine residues (SEQ ID NO:23).

In some embodiments of the ninth and tenth aspects of the invention, the CD38 binding domain is linked to the Fc domain monomer by a linker. In some embodiments, the linker is a spacer.

In some embodiments of the ninth and tenth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position 1253. In some embodiments, each amino acid modification at position I253 is independently I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y. In some embodiments, each amino acid modification at position 1253 is 1253A.

In some embodiments of the ninth and tenth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position R292. In some embodiments, each amino acid modification at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid modification at position R292 is R292P.

In some embodiments of the ninth and tenth aspects of the invention, one or more of the Fc domain monomers comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, each Fc domain monomer comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, the IgG has a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

In some embodiments of the ninth and tenth aspects of the invention, the N-terminal Asp in each of the first, second, third, and fourth polypeptides is mutated to Gln.

In some embodiments of the ninth and tenth aspects of the invention, at least one of the first, second, third, and fourth polypeptides lacks a C-terminal lysine. In some embodiments, each of the first, second, third, and fourth polypeptides lacks a C-terminal lysine.

In some embodiments of the ninth and tenth aspects of the invention, the Fc-antigen binding domain construct further comprises an albumin-binding peptide linked to the N-terminus or C-terminus of one or more of the polypeptides by a linker. .

In some embodiments of the eleventh aspect of the invention, the first Fc domain monomer and the third Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer. wherein the second Fc domain monomer and the fourth Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fourth Fc domain monomer, wherein the second and third polypeptides are have different amino acid sequences.

In some embodiments of the eleventh aspect of the invention, the first CD38 binding domain is linked to the first polypeptide and the second CD38 binding domain is linked to the second polypeptide and the third polypeptide.

In some embodiments of the eleventh aspect of the invention, each of the second Fc domain monomer and the fourth Fc domain monomer comprises E357K and K370D, and each of the first Fc domain monomer and the third Fc domain monomer comprises K370D and E357K includes

In some embodiments of the twelfth aspect of the invention, the first Fc domain monomer and the third Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer. wherein the second Fc domain monomer and the fourth Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fourth Fc domain monomer, wherein the second and third polypeptides are have different amino acid sequences.

In some embodiments of the twelfth aspect of the invention, each of the second Fc domain monomer and the fourth Fc domain monomer comprises D399K and K409D, and each of the first Fc domain monomer and the third Fc domain monomer is E357K and K370D includes

In some embodiments of the eleventh and twelfth aspects of the invention, the first or CD38 binding domain is a Fab or V _H domain. In some embodiments of the eleventh and twelfth aspects of the invention, the first and second CD38 binding domains are Fab. In some embodiments of the ninth aspect of the invention, the first, second, and third CD38 binding domains are Fab or V _H domains.

In some embodiments of the eleventh and twelfth aspects of the invention, the first or second CD38 binding domain is an scFv. In some embodiments of the eleventh and twelfth aspects of the invention, the first and second CD38 binding domains are scFvs. In some embodiments of the ninth aspect of the invention, the first, second, and third CD38 binding domains are scFvs.

In some embodiments of the eleventh aspect of the invention, the first or second CD38 domain comprises a V _H domain and a C _H 1 domain, wherein the V _H and C _H 1 domains are the first, second, or third polypeptides. It is part of the amino acid sequence of In some embodiments, the CD38 binding domain further comprises a _VL domain, and in some embodiments the Fc-antigen binding domain construct comprises a fourth polypeptide comprising a _VL domain. In some embodiments, the V _H domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, or the V _H domain comprises a CDR of a V _H domain comprising the sequences of an antibody set forth in Table 2 -H1, CDR-H2, and CDR-H3, or the V _H domain comprises CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody set forth in Table 2, wherein the CDR-H1, CDR the V _H sequence excluding the -H2 and CDR-H3 sequences is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H sequence of the antibody set forth in Table 2, or The V _H domain comprises the V _H sequence of an antibody shown in Table 2.

In some embodiments of the twelfth aspect of the invention, the first, second, or third CD38 binding domain comprises a V _H domain and a C _H 1 domain, wherein the V _H and C _H 1 domains comprise the first, second , or part of the amino acid sequence of a third polypeptide. In some embodiments, the CD38 binding domain further comprises a _VL domain, and in some embodiments the Fc-antigen binding domain construct comprises a fourth polypeptide comprising a _VL domain. In some embodiments, the V _H domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, or the V _H domain comprises a CDR of a V _H domain comprising the sequences of an antibody set forth in Table 2 -H1, CDR-H2, and CDR-H3, or the V _H domain comprises CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody set forth in Table 2, wherein the CDR-H1, CDR the V _H sequence excluding the -H2 and CDR-H3 sequences is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H sequence of the antibody set forth in Table 2, or The V _H domain comprises the V _H sequence of an antibody shown in Table 2.

In some embodiments of the eleventh aspect of the invention, the first or second CD38 binding domain comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences set forth in Table 1 or the CD38 binding domain is CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 from the set of V _H and V _L sequences of an antibody shown in Table 2. sequence, or the CD38 binding domain comprises a V _H domain comprising the CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of the antibody shown in Table 2, and the CDR of the _VL sequence of the antibody shown in Table 2 -V _H comprising a V _L domain comprising -L1, CDR-L2, and CDR-L3, and excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences and the V _L domain sequence is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H and V _L sequences of the antibody set forth in Table 2, or the CD38 binding domain is 2 contains the set of V _H and V _L sequences of the antibody.

In some embodiments of the twelfth aspect of the invention, the first, second, or third CD38 binding domain comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and comprising the set of CDR-L3 sequences, or the CD38 binding domain comprises CDR- _H1 , CDR-H2, CDR-H3, CDR- _L1 , CDR-L2, and a CDR-L3 sequence, or the CD38 binding domain comprises a V H domain comprising the CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of the antibody set forth in Table 2, and the V _H domain of the antibody set forth in Table 2 a _VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the _L sequence, and a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequence the V _H and V _L domain sequences except for are at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H and V _L sequences of the antibodies set forth in Table 2, or CD38 The binding domain comprises the set of V _H and V _L sequences of an antibody shown in Table 2.

In some embodiments of the eleventh and twelfth aspects of the invention, the Fc-antigen binding domain construct further comprises an IgG C _L antibody constant domain and an IgG C _H 1 antibody constant domain, wherein the Fc-antigen binding domain construct further comprises an IgG C _H 1 antibody constant domain. The domain is attached to the N-terminus of the first polypeptide or the second polypeptide by a linker.

In some embodiments of the eleventh and twelfth aspects of the invention, the first Fc domain monomer and the third Fc domain monomer undergo complementary dimerization to promote dimerization between the first Fc domain monomer and the third Fc domain monomer. Includes a selectivity module.

In some embodiments of the eleventh and twelfth aspects of the invention, the second Fc domain monomer and the fourth Fc domain monomer undergo complementary dimerization to promote dimerization between the second Fc domain monomer and the fourth Fc domain monomer. Includes a selectivity module.

In some embodiments of the eleventh and twelfth aspects of the invention, the dimerization selectivity module comprises an engineered cavity into the C _H 3 domain of one of the Fc domain monomers and the C _H 3 domain of the other of the Fc domain monomers. and an engineered protrusion into the cavity, wherein the engineered cavity and the engineered protrusion are positioned to form a protrusion pair into the cavity of the Fc domain monomers. In some embodiments, the engineered protrusion comprises at least one modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and wherein the engineered cavity comprises Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and at least one modification selected from T394S. In some embodiments, one of the Fc domain monomers comprises Y407V and Y349C and the other of the Fc domain monomers comprises T366W and S354C.

In some embodiments of the eleventh and twelfth aspects of the invention, the dimerization selectivity module comprises a negatively charged amino acid into the C _H 3 domain of one of the domain monomers and C _H 3 of the other of the Fc domain monomers. positively charged amino acids into the domain, wherein the negatively and positively charged amino acids are positioned to promote formation of the Fc domain. In some embodiments, each of the first and third Fc domain monomers comprises D399K and either K409D or K409E, or each of the first and third Fc domain monomers comprises K392D and D399K or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370E, or each of the first Fc domain monomer and the third Fc domain monomer comprises D356K and K439D, or each of the first Fc domain the monomer and the third Fc domain monomer comprise K392E and D399K, or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370D, or each of the first Fc domain monomer and the third Fc domain monomer comprises: D356K and K439E, or each of the second and fourth Fc domain monomers comprises S354C and T366W and each of the third and fourth polypeptides comprises Y349C, T366S, L368A, and Y407V, respectively wherein the third and fourth polypeptides comprise S354C and T366W and each of the second Fc domain monomer and the fourth Fc domain monomer comprises Y349C, T366S, L368A, and Y407V, or each of the second Fc domain monomer and the first the 4 Fc domain monomers comprise E357K or E357R and each of the third and fourth polypeptides comprises K370D or K370E, or each of the second and fourth Fc domain monomers comprises K370D or K370E and each the third and fourth polypeptides comprise E357K or 357R, or each of the second and fourth Fc domain monomers comprises K409D or K409E and each of the third and fourth polypeptides comprises D399K or D399R or each of the second Fc domain monomer and the fourth F The c domain monomer comprises D399K or D399R and each of the third and fourth polypeptides comprises K409D or K409E.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a bond.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a spacer. 일부 실시 형태에서, 스페이서는 서열 GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGG. In some embodiments, the spacer is a glycine spacer, for example consisting of 4-30, 8-30, or 12-30 glycine residues, such as a spacer consisting of 20 glycine residues.

In some embodiments of the eleventh and twelfth aspects of the invention, one or more of the CD38 binding domains are linked to the Fc domain monomer by a linker. In some embodiments, the linker is a spacer.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position 1253. In some embodiments, each amino acid modification at position I253 is independently I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y. In some embodiments, each amino acid modification at position 1253 is 1253A.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position R292. In some embodiments, each amino acid modification at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid modification at position R292 is R292P.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one of the Fc domain monomers comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, each Fc domain monomer comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, the IgG has a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

In some embodiments of the eleventh and twelfth aspects of the invention, the N-terminal Asp in each of the first, second, third, and fourth polypeptides is mutated to Gln.

In some embodiments of the eleventh and twelfth aspects of the invention, at least one of the first, second, third, and fourth polypeptides lacks a C-terminal lysine. In some embodiments, each of the first, second, third, and fourth polypeptides lacks a C-terminal lysine.

In some embodiments of the eleventh and twelfth aspects of the invention, the Fc-antigen binding domain construct further comprises an albumin-binding peptide linked to the N-terminus or C-terminus of one or more of the polypeptides by a linker. .

In a thirteenth aspect, the invention features a composition comprising a substantially homogeneous population of Fc-antigen binding domain constructs, said Fc-antigen binding domain construct comprising: a) i) a first Fc domain monomer, ii ) a second Fc domain monomer, and iii) a first polypeptide comprising a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising i) a third Fc domain monomer, ii) a fourth Fc domain monomer, and iv) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and d) a CD38 binding domain linked to said first polypeptide, second polypeptide, third polypeptide, or fourth polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain and the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain .

In some embodiments of the thirteenth aspect of the invention, each of the first Fc domain monomer and the third Fc domain monomer is a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer. wherein each of the second Fc domain monomer and the fifth Fc domain monomer comprises a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fifth Fc domain monomer; The Fc domain monomer and the sixth Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the fourth and sixth Fc domain monomers.

In a fourteenth aspect, the invention features a composition comprising a substantially homogeneous population of Fc-antigen binding domain constructs, said Fc-antigen binding domain construct comprising: a) i) a first Fc domain monomer, ii ) a second Fc domain monomer, and iii) a first polypeptide comprising a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising i) a third Fc domain monomer, ii) a fourth Fc domain monomer, and iv) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and e) a CD38 binding domain linked to said first, second, third, or fourth polypeptide, wherein said second Fc domain monomer and said fourth Fc domain monomer combine to form a first Fc domain and the first Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain .

In some embodiments of the fourteenth aspect of the invention, each of the second Fc domain monomer and the fourth Fc domain monomer is a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fourth Fc domain monomer. wherein each of the first Fc domain monomer and the fifth Fc domain monomer comprises a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the fifth Fc domain monomer, wherein each third The Fc domain monomer and the sixth Fc domain monomer comprise a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the sixth Fc domain monomer.

In a fifteenth aspect, the invention features a composition comprising a substantially homogeneous population of Fc-antigen binding domain constructs, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii ) a second Fc domain monomer, iii) a third Fc domain monomer, iv) a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and v) a first polypeptide comprising a second linker connecting the second Fc domain monomer and the third Fc domain monomer; b) i) a fourth Fc domain monomer, ii) a fifth Fc domain monomer, iii) a sixth Fc domain monomer, iv) a third linker connecting the fourth Fc domain monomer and the fifth Fc domain monomer; and v) a second polypeptide comprising a fourth linker connecting the fifth Fc domain monomer and the sixth Fc domain monomer; c) a third polypeptide comprising a seventh Fc domain monomer; d) a fourth polypeptide comprising an eighth Fc domain monomer; e) a fifth polypeptide comprising a ninth Fc domain monomer; f) a sixth polypeptide comprising a tenth Fc domain monomer; and g) a CD38 binding domain linked to a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, or a sixth polypeptide, wherein the second Fc domain monomer and the fifth Fc domain monomer are combining to form a first Fc domain, combining the first Fc domain monomer and the seventh Fc domain monomer to form a second Fc domain, and combining the fourth Fc domain monomer and the eighth Fc domain monomer a third Fc domain is formed, the third Fc domain monomer and the ninth Fc domain monomer are combined to form a fourth Fc domain, and the sixth Fc domain monomer and the tenth Fc domain monomer are combined to form a fifth form the Fc domain.

In some embodiments of the fifteenth aspect of the invention, each of the second domain monomer and the fifth Fc domain monomer has a complementary dimerization selectivity that promotes dimerization between the second and fifth Fc domain monomers. module, wherein each of the first Fc domain monomer and the seventh Fc domain monomer comprises a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the seventh Fc domain monomer, each The fourth Fc domain monomer and the eighth Fc domain monomer of and the ninth Fc domain monomer comprises a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the ninth Fc domain monomer, each of the sixth Fc domain monomer and the tenth Fc domain monomer comprises a complementary dimerization selectivity module that promotes dimerization between the sixth Fc domain monomer and the tenth Fc domain monomer.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the CD38 binding domain is a Fab or V _H domain.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the CD38 binding domain is part of the amino acid sequence of one or more of the polypeptides, and in some embodiments, the CD38 binding domain is an scFv.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the CD38 binding domain comprises a V _H domain and a C _H 1 domain, wherein the V _H and C _H 1 domains comprise the first, second , or part of the amino acid sequence of a third polypeptide. In some embodiments, the CD38 binding domain further comprises a _VL domain, and in some embodiments the Fc-antigen binding domain construct comprises a fourth polypeptide comprising a _VL domain. In some embodiments, the V _H domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, or the V _H domain comprises a CDR of a V _H domain comprising the sequences of an antibody set forth in Table 2 -H1, CDR-H2, and CDR-H3, or the V _H domain comprises CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of an antibody set forth in Table 2, wherein the CDR-H1, CDR the V _H sequence excluding the -H2 and CDR-H3 sequences is at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H sequence of the antibody set forth in Table 2, or The V _H domain comprises the V _H sequence of an antibody shown in Table 2.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the CD38 binding domain comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and comprising the set of CDR-L3 sequences, or the CD38 binding domain comprises CDR- _H1 , CDR-H2, CDR-H3, CDR- _L1 , CDR-L2, and a CDR-L3 sequence, or the CD38 binding domain comprises a V H domain comprising the CDR-H1, CDR-H2, and CDR-H3 of the V _H sequence of the antibody set forth in Table 2, and the V _H domain of the antibody set forth in Table 2 a _VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the _L sequence, and a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequence the V _H and V _L domain sequences except for are at least 95% identical, at least 97% identical, at least 99% identical, or at least 99.5% identical to the V _H and V _L sequences of the antibodies set forth in Table 2, or CD38 The binding domain comprises the set of V _H and V _L sequences of an antibody shown in Table 2.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the Fc-antigen binding domain construct further comprises an IgG C _L antibody constant domain and an IgG C _H 1 antibody constant domain, The C _H 1 antibody constant domain is attached to the N-terminus of the first polypeptide or the second polypeptide by a linker.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the dimerization selectivity module is an engineered cavity into the C _H 3 domain of one of the Fc domain monomers and the other of the Fc domain monomers. comprising an engineered protrusion into the C _H 3 domain of the engineered cavity and the engineered protrusion positioned to form a protrusion pair into the cavity of the Fc domain monomers. In some embodiments, the engineered protrusion comprises at least one modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and wherein the engineered cavity comprises Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and at least one modification selected from T394S. In some embodiments, one of the Fc domain monomers comprises Y407V and Y349C and the other of the Fc domain monomers comprises T366W and S354C.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the dimerization selectivity module comprises a negatively charged amino acid into the C _H 3 domain of one of the domain monomers and the other of the Fc domain monomers. positively charged amino acids into one C _H 3 domain, wherein the negatively and positively charged amino acids are positioned to promote formation of the Fc domain. In some embodiments, each of the first and third Fc domain monomers comprises D399K and either K409D or K409E, or each of the first and third Fc domain monomers comprises K392D and D399K or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370E, or each of the first Fc domain monomer and the third Fc domain monomer comprises D356K and K439D, or each of the first Fc domain the monomer and the third Fc domain monomer comprise K392E and D399K, or each of the first Fc domain monomer and the third Fc domain monomer comprises E357K and K370D, or each of the first Fc domain monomer and the third Fc domain monomer comprises: D356K and K439E, or each of the second and fourth Fc domain monomers comprises S354C and T366W and each of the third and fourth polypeptides comprises Y349C, T366S, L368A, and Y407V, respectively wherein the third and fourth polypeptides comprise S354C and T366W and each of the second Fc domain monomer and the fourth Fc domain monomer comprises Y349C, T366S, L368A, and Y407V, or each of the second Fc domain monomer and the first the 4 Fc domain monomers comprise E357K or E357R and each of the third and fourth polypeptides comprises K370D or K370E, or each of the second and fourth Fc domain monomers comprises K370D or K370E and each the third and fourth polypeptides comprise E357K or 357R, or each of the second and fourth Fc domain monomers comprises K409D or K409E and each of the third and fourth polypeptides comprises D399K or D399R or each of the second Fc domain monomer and the fourth F The c domain monomer comprises D399K or D399R and each of the third and fourth polypeptides comprises K409D or K409E.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the one or more linkers in the Fc-antigen binding domain construct is a bond.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, at least one linker in the Fc-antigen binding domain construct is a spacer. 일부 실시 형태에서, 스페이서는 서열 GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGG. In some embodiments, the spacer is a glycine spacer, for example consisting of 4-30, 8-30, or 12-30 glycine residues, such as a spacer consisting of 20 glycine residues.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the CD38 binding domain is linked to the Fc domain monomer by a linker. In some embodiments, the linker is a spacer.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position 1253. In some embodiments, each amino acid modification at position I253 is independently I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y. In some embodiments, each amino acid modification at position 1253 is 1253A.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, at least one of the Fc domains comprises at least one amino acid modification at position R292. In some embodiments, each amino acid modification at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid modification at position R292 is R292P.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, at least one of the Fc domain monomers comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. include In some embodiments, each Fc domain monomer comprises an IgG hinge domain, an IgG C _H 2 antibody constant domain, and an IgG C _H 3 antibody constant domain. In some embodiments, the IgG has a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the N-terminal Asp in each polypeptide is mutated to Gin.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, at least one of the polypeptides lacks a C-terminal lysine. In some embodiments, each polypeptide lacks a C-terminal lysine.

In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of the invention, the Fc-antigen binding domain construct comprises an albumin-binding peptide linked to the N-terminus or C-terminus of one or more of the polypeptides by a linker. further includes.

In a 16 aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer; b) a second polypeptide comprising a third Fc domain monomer; c) a third polypeptide comprising a fourth Fc domain monomer; and d) a first CD38 binding domain linked to said first polypeptide; and e) a second CD38 binding domain linked to said second and/or third polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain, 2 Fc domain monomers and said fourth Fc domain monomer combine to form a second Fc domain, wherein said first CD38 binding domain and said second CD38 binding domain bind different antigens, and wherein said Fc-antigen binding domain construct improved effector function compared to constructs with a single Fc domain and a CD38 binding domain in antibody-dependent cytotoxicity (ADCC) assays, antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assays has

In a 26 aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising iv) a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and d) a CD38 binding domain linked to said first polypeptide, second polypeptide, third polypeptide, or fourth polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain forming, wherein the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain; , wherein said Fc-antigen binding domain construct comprises a single Fc domain and a CD38 binding domain in antibody-dependent cytotoxicity (ADCC) assays, antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assays. It has an improved effector function compared to the construct with

In a 27th aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising iv) a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and e) a CD38 binding domain linked to said first, second, third, or fourth polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain forming, wherein the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain; , wherein said Fc-antigen binding domain construct comprises a single Fc domain and a biological activity not exhibited by the construct with said CD38 binding domain.

In a 28 aspect, the invention features an Fc-antigen binding domain construct, wherein said Fc-antigen binding domain construct comprises a) i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) a first polypeptide comprising a first spacer connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising iv) a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second spacer connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and e) a CD38 binding domain linked to said first, second, third, or fourth polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain and the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain .

In a 29 aspect, the invention features a cell culture medium comprising a population of Fc-antigen binding domain constructs, wherein at least 50% of said Fc-antigen binding domain constructs on a molar basis comprise a) i) a first a first polypeptide comprising an Fc domain monomer, ii) a second Fc domain monomer, and iii) a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and b) a second polypeptide comprising iv) a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and c) a third polypeptide comprising a fifth Fc domain monomer; d) a fourth polypeptide comprising a sixth Fc domain monomer; and e) a CD38 binding domain linked to said first, second, third, or fourth polypeptide, wherein said first Fc domain monomer and said third Fc domain monomer combine to form a first Fc domain and the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain .

In a thirtieth aspect, the invention features a method of making an Fc-antigen binding domain construct, said method comprising: a) (1) (i) a first Fc domain monomer, ii) a second Fc domain monomer, and iii) ) a first polypeptide comprising a first linker connecting the first Fc domain monomer and the second Fc domain monomer; and (2) a second polypeptide comprising (2) a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second linker connecting the third Fc domain monomer and the fourth Fc domain monomer; and (3) a third polypeptide comprising a fifth Fc domain monomer; (4) a fourth polypeptide comprising a sixth Fc domain monomer; and (5) culturing a host cell expressing a CD38 binding domain linked to the first, second, third, or fourth polypeptide, wherein the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain, the second Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the fourth Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain, wherein at least 50% of the Fc-antigen binding domain construct in the cell culture supernatant on a molar basis is structurally identical, and b) the Fc-antigen from the cell culture supernatant purifying the binding domain construct.

In some embodiments of the 26th, 27th, 28th, 29th, and 30th aspects of the invention, each of the first Fc domain monomer and the third Fc domain monomer comprises the first Fc domain monomer and the second a complementary dimerization selectivity module that promotes dimerization between 3 Fc domain monomers, wherein each of the second Fc domain monomer and the fifth Fc domain monomer comprises a dimerization between the second Fc domain monomer and the fifth Fc domain monomer. a complementarity dimerization selectivity module for promoting contains modules.

In some embodiments of all aspects of the invention, the Fc-antigen binding domain construct has reduced fucosylation. Thus, in some embodiments, less than 40%, 30%, 20%, 15%, 10% or 5% of the Fc domain monomers in a composition comprising the Fc-antigen binding domain construct are fucosylated.

In some embodiments of all aspects of the invention, the Fc domain monomers of Figure 24A have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid changes in the CH3 domain. amino acid sequence (SEQ ID NO: 43).

In some embodiments of all aspects of the invention, the Fc domain monomers of Figure 24B have up to 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid changes in the CH3 domain. amino acid sequence (SEQ ID NO: 45).

In some embodiments of all aspects of the invention, the Fc domain monomers have at most 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid changes in the CH3 domain of Figure 24C. amino acid sequence (SEQ ID NO: 47).

In some embodiments of all aspects of the invention, the Fc domain monomers have at most 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid changes in the CH3 domain of Figure 24D. amino acid sequence (SEQ ID NO: 42).

In some embodiments of all aspects of the invention, the Fc domain monomer does not comprise K447, for example when the Fc domain monomer is at the carboxy-terminal end of the polypeptide. In other embodiments, the Fc domain monomer comprises K447, eg, when the Fc domain monomer is not present at the carboxy-terminal end of the polypeptide.

In some embodiments of all aspects of the invention, for example, when the Fc domain monomer is amino-terminal to the linker, the Fc domain monomer does not comprise a portion of the hinge of E216 to C220 (including the endpoints), but D221 to L235 (including endpoints). In other embodiments, for example, when the Fc domain monomer is carboxy-terminally to the CH1 domain, the Fc domain monomer comprises a portion of the hinge from E216 to L235, inclusive. In some embodiments of all aspects of the invention, the hinge domain, eg the hinge domain at the amino-terminus of the polypeptide, has an Asp to Gln mutation at EU position 221.

As mentioned above, the Fc-antigen binding domain constructs of the invention are assembled from polypeptides, comprising polypeptides comprising two or more IgG1 Fc domain monomers, and such polypeptides are an aspect of the invention.

In a forty-first aspect, the invention features a polypeptide, said polypeptide comprising a CD38 binding domain; linker; a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein at least one Fc domain monomer comprises a mutation that forms an engineered protrusion.

In various embodiments of the 41st aspect, the CD38 binding domain comprises an antibody heavy chain variable domain; the CD38 binding domain comprises an antibody light chain variable domain; the first IgGl Fc domain monomer comprises two or four reverse charge mutations and the second IgGl Fc domain monomer comprises a mutation that forms an engineered protrusion; the first IgGl Fc domain monomer comprises a mutation forming an engineered protrusion and the second IgGl Fc domain monomer comprises 2 or 4 charge reversal mutations; both the first IgG1 Fc domain monomer and the second IgG constant domain monomer comprise mutations that form engineered protrusions; the polypeptide comprises a third linker and a third IgG1 Fc domain monomer, wherein each of the first IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer comprises a mutation that forms an engineered protrusion; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the first IgGl Fc domain monomer and the second IgGl Fc domain monomer each comprise a mutation that forms an engineered protrusion, and wherein the third IgGl Fc domain The monomer contains 2 or 4 charge inversion mutations; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the first IgGl Fc domain monomer and the third IgGl Fc domain monomer each comprise a mutation forming an engineered protrusion, and wherein the second IgGl domain monomer contains 2 or 4 charge inversion mutations; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the second IgGl Fc domain monomer and the third IgGl Fc domain monomer each comprise a mutation that forms an engineered protrusion, and wherein the first IgGl domain monomer contains 2 or 4 charge inversion mutations.

In various embodiments of the 41st aspect, the IgG1 Fc domain monomer comprising the engineered protrusion forming mutation further comprises 1, 2 or 3 charge reversal mutations; Mutations that form engineered protrusions and charge inversion mutations are in the CH3 domain; The mutation is in the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); A mutation is a single amino acid change; The second linker and the optional third linker are GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, RGSGGS, RGSGGS, RGS, RGP, GGS, GGS, GGS, GGSG, GGGS, GGSG, GGSG, GGSG, GSGS, GSGS, GSGS, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGGGGGGG and selected from the group consisting of: the second linker and optional third linker are glycine spacers; the second linker and the optional third linker independently consist of 4-30, 4-20, 8-30, 8-20, 12-20 or 12-30 glycine residues; the second linker and optional third linker consist of 20 glycine residues; At least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position I253, wherein each amino acid mutation at EU numbering position I253 is independently 1253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I , I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y; each amino acid mutation at position 1253 is 1253A; At least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position R292, wherein each amino acid mutation at EU numbering position R292 is independently R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and selected from the group consisting of R292Y; each amino acid mutation at position R292 is R292P; each Fc domain monomer independently comprises or consists of an amino acid sequence selected from the group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge portion of the second Fc domain monomer and the third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL, and the hinge portion of the second Fc domain monomer and the third Fc domain monomer has the amino acid sequence DKTHTCPPCPAPELL; the CH2 domain of each Fc domain monomer independently comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid substitutions; The CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid substitutions; The single amino acid substitution consists of T366Y, T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R. selected from the group; each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions; up to 6 of the single amino acid substitutions are charge inversion mutations in the CH3 domain or mutations that form engineered protrusions; the single amino acid substitution is within the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); at least one of the mutations forming the engineered protrusion is selected from the group consisting of T366Y, T366W, T394W, T394Y, F405W, S354C, Y349T, and T394F; Two or four charge inversion mutations are K409D, K409E, K392D. K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; the CD38 binding domain is an scFv; the CD38 binding domain comprises a VH domain and a CH1 domain; the CD38 binding domain further comprises a VL domain; The VH domain comprises the set of CDR-H1, CDR-H2 and CDR-H3 sequences shown in Table 1; the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 of a VH domain comprising the sequence of an antibody shown in Table 2; The VH domain comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH sequences of the antibody shown in Table 2, and the VH sequences excluding the CDR-H1, CDR-H2, and CDR-H3 sequences are shown in Table 2 at least 95% or 98% identical to the VH sequence of the antibody; The VH domain comprises the VH sequence of an antibody shown in Table 2; The CD38 binding domain comprises the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences set forth in Table 1; the CD38 binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from the set of VH and VL sequences of an antibody shown in Table 2; The CD38 binding domain comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of the VH sequences of the antibody shown in Table 2, and CDR-L1, CDR-L2, and CDR-L2 of the VL sequence of the antibody shown in Table 2; An antibody comprising a VL domain comprising CDR-L3, wherein the VH and VL domain sequences excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences are shown in Table 2 at least 95% or 98% identical to the VH and VL sequences of The CD38 binding domain comprises the set of VH and VL sequences of an antibody shown in Table 2; the CD38 binding domain comprises an IgG CL antibody constant domain and an IgG CH1 antibody constant domain; The CD38 binding domain comprises a VH domain and a CH1 domain and is capable of binding to a polypeptide comprising a VL domain and a CL domain to form a Fab.

Also described are polypeptide complexes comprising two copies of the aforementioned polypeptides linked by disulfide bonds between cysteine residues in the hinge of a first or second IgG1 Fc domain monomer.

Also described is a polypeptide complex comprising a polypeptide as described above linked to a second polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the polypeptide and the second polypeptide comprise a first, a first polypeptide of the polypeptide. are linked by a disulfide bond between the hinge domain of the second or third IgG1 Fc domain monomer and the cysteine residues in the hinge domain of the second polypeptide.

In various embodiments of the complex, the second polypeptide monomer comprises a mutation that forms an engineered cavity; the mutation forming the engineered cavity is selected from the group consisting of Y407T, Y407A, F405A, T394S, T394W/Y407A, T366W/T394S, T366S/L368A/Y407V/Y349C, S364H/F405A; The second polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions.

In a forty-two aspect, the invention features a polypeptide, said polypeptide comprising a CD38 binding domain; linker; a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the at least one Fc domain monomer comprises 1, 2 or 3 charge inversion amino acid mutations.

In various embodiments of the 42nd aspect, the CD38 binding domain comprises an antibody heavy chain variable domain; the CD38 binding domain comprises an antibody light chain variable domain; The first IgG1 Fc domain monomer comprises a set of two charge reversal mutations selected from those set forth in Tables 4A and 4B or a set of four charge reversal mutations selected from those set forth in Tables 4A and 4B, and a second the IgG1 Fc domain monomer comprises 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B; The first IgG1 Fc domain monomer comprises 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B, and the second IgG1 Fc domain monomer has two charges selected from those in Tables 4A and 4B. a set of inversion mutations or a set of four charge inversion mutations selected from those in Tables 4A and 4B; both the first IgG1 Fc domain monomer and the second IgG constant domain monomer comprise 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein each of the first IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer is selected from Table 4A and Table 4B. , comprising two or three charge-reversing amino acid mutations; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the first IgG1 Fc domain monomer and the second IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively and wherein the third IgG1 Fc domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversions selected from those in Tables 4A and 4B. comprising a set of mutations; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the first IgG1 Fc domain monomer and the third IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively and wherein the second IgG1 domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversion mutations selected from those in Tables 4A and 4B. contains a set of; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively wherein the first IgG1 domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversion mutations selected from those in Tables 4A and 4B. contains a set of; IgGl Fc domain monomers comprising 1, 2 or 3 charge inversion amino acid mutations selected from Tables 4A and 4B have the same CH3 domain; 1, 2 or 3 charge inversion amino acid mutations selected from Tables 4A and 4B are present in the CH3 domain; The mutation is in the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); Each mutation is a single amino acid change; The mutation is in the sequence from EU numbering position G341 to EU numbering position K446 (inclusive); A mutation is a single amino acid change; The second linker and the optional third linker are GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, RGSGGS, RGSGGS, RGS, RGP, GGS, GGS, GGS, GGSG, GGGS, GGSG, GGSG, GGSG, GSGS, GSGS, GSGS, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGGGGGGG and selected from the group consisting of: the second linker and optional third linker are glycine spacers; the second linker and the optional third linker independently consist of 4-30, 4-20, 8-30, 8-20, 12-20 or 12-30 glycine residues; the second linker and optional third linker consist of 20 glycine residues; At least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position I253, wherein each amino acid mutation at EU numbering position I253 is independently 1253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I , I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y; each amino acid mutation at position 1253 is 1253A; at least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position R292; each amino acid mutation at EU numbering position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is R292P; each Fc domain monomer independently comprises or consists of an amino acid sequence selected from the group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge portion of the second Fc domain monomer and the third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL, and the hinge portion of the second Fc domain monomer and the third Fc domain monomer has the amino acid sequence DKTHTCPPCPAPELL; the CH2 domain of each Fc domain monomer independently comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid substitutions; The CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid substitutions; The single amino acid substitution consists of T366Y, T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R. selected from the group; each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions; up to 6 of the single amino acid substitutions are charge inversion mutations in the CH3 domain or mutations that form engineered protrusions; the single amino acid substitution is within the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); at least one of the mutations forming the engineered protrusion is selected from the group consisting of T366Y, T366W, T394W, T394Y, F405W, S354C, Y349T, and T394F; Two or four charge inversion mutations are K409D, K409E, K392D. K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; the CD38 binding domain is an scFv; the CD38 binding domain comprises a VH domain and a CH1 domain; the CD38 binding domain further comprises a VL domain; The VH domain comprises the set of CDR-H1, CDR-H2 and CDR-H3 sequences shown in Table 1; the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 of a VH domain comprising the sequence of an antibody shown in Table 2; The VH domain comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH sequences of the antibody shown in Table 2, and the VH sequences excluding the CDR-H1, CDR-H2, and CDR-H3 sequences are shown in Table 2 at least 95% or 98% identical to the VH sequence of the antibody; The VH domain comprises the VH sequence of an antibody shown in Table 2; The CD38 binding domain comprises the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences set forth in Table 1; the CD38 binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from the set of VH and VL sequences of an antibody shown in Table 2; The CD38 binding domain comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of the VH sequences of the antibody shown in Table 2, and CDR-L1, CDR-L2, and CDR-L2 of the VL sequence of the antibody shown in Table 2; An antibody comprising a VL domain comprising CDR-L3, wherein the VH and VL domain sequences excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences are shown in Table 2 at least 95% or 98% identical to the VH and VL sequences of The CD38 binding domain comprises the set of VH and VL sequences of an antibody shown in Table 2; the CD38 binding domain comprises an IgG CL antibody constant domain and an IgG CH1 antibody constant domain; The CD38 binding domain comprises a VH domain and a CH1 domain and is capable of binding to a polypeptide comprising a VL domain and a CL domain to form a Fab.

Also described are polypeptide complexes comprising two copies of any of the foregoing polypeptides linked by disulfide bonds between cysteine residues in the hinge of a first or second IgG1 Fc domain monomer.

Also described is a polypeptide complex comprising a polypeptide as described above linked to a second polypeptide comprising an IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the polypeptide and the second polypeptide comprise a first, a first polypeptide of the polypeptide. are linked by a disulfide bond between the hinge domain of the second or third IgG1 Fc domain monomer and the cysteine residues in the hinge domain of the second polypeptide. In various embodiments, the second polypeptide monomer comprises 1, 2, or 3 charge inversion mutations; the second polypeptide monomer comprises 1, 2 or 3 charge reversal mutations selected from Tables 4A and 4B and is complementary to 1, 2 or 3 charge reversal mutations selected from Tables 4A and 4B in the polypeptide; The second polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions.

In a 43 aspect, the invention features a polypeptide comprising a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein at least one Fc domain monomer comprises a mutation that forms an engineered protrusion.

In various embodiments of the 43 aspect, the polypeptide further comprises an antibody heavy chain variable domain, and a CH1 domain amino-terminal to the first IgG1 monomer or an scFv amino-terminal to the first IgG1 monomer; the first IgG1 Fc domain monomer comprises two or four charge inversion mutations and the second IgG1 Fc domain monomer comprises a mutation that forms an engineered protrusion; the first IgGl Fc domain monomer comprises a mutation forming an engineered protrusion and the second IgGl Fc domain monomer comprises 2 or 4 charge reversal mutations; both the first IgG1 Fc domain monomer and the second IgG constant domain monomer comprise mutations that form engineered protrusions; the polypeptide comprises a third linker and a third IgG1 Fc domain monomer, wherein each of the first IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer comprises a mutation that forms an engineered protrusion; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the first IgGl Fc domain monomer and the second IgGl Fc domain monomer each comprise a mutation that forms an engineered protrusion, and wherein the third IgGl Fc domain The monomer contains 2 or 4 charge inversion mutations; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the first IgGl Fc domain monomer and the third IgGl Fc domain monomer each comprise a mutation forming an engineered protrusion, and wherein the second IgGl domain monomer contains 2 or 4 charge inversion mutations; The polypeptide comprises a third linker and a third IgGl Fc domain monomer, wherein both the second IgGl Fc domain monomer and the third IgGl Fc domain monomer each comprise a mutation that forms an engineered protrusion, and wherein the first IgGl domain monomer contains 2 or 4 charge inversion mutations.

In various embodiments of the 43 aspect, the IgG1 Fc domain monomer comprising the engineered protrusion forming mutation further comprises 1, 2 or 3 charge inversion mutations;

Mutations that form engineered protrusions and charge inversion mutations are present in the CH3 domain; The mutation is in the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); A mutation is a single amino acid change; The second linker and the optional third linker are GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, RGSGGS, RGSGGS, RGS, RGP, GGS, GGS, GGS, GGSG, GGGS, GGSG, GGSG, GGSG, GSGS, GSGS, GSGS, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGGGGGGG and selected from the group consisting of: the second linker and optional third linker are glycine spacers; the second linker and the optional third linker independently consist of 4-30, 4-20, 8-30, 8-20, 12-20 or 12-30 glycine residues; the second linker and optional third linker consist of 20 glycine residues; At least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position I253, wherein each amino acid mutation at EU numbering position I253 is independently 1253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I , I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y; each amino acid mutation at position 1253 is 1253A; at least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position R292; each amino acid mutation at EU numbering position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is R292P; each Fc domain monomer independently comprises or consists of an amino acid sequence selected from the group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge portion of the second Fc domain monomer and the third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL, and the hinge portion of the second Fc domain monomer and the third Fc domain monomer has the amino acid sequence DKTHTCPPCPAPELL; the CH2 domain of each Fc domain monomer independently comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid substitutions; The CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid substitutions; The single amino acid substitution consists of T366Y, T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R. selected from the group; each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions; up to 6 of the single amino acid substitutions are charge inversion mutations in the CH3 domain or mutations that form engineered protrusions; the single amino acid substitution is within the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); at least one of the mutations forming the engineered protrusion is selected from the group consisting of T366Y, T366W, T394W, T394Y, F405W, S354C, Y349T, and T394F; Two or four charge inversion mutations are K409D, K409E, K392D. K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R.

In a 44 aspect, the invention features a polypeptide comprising a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an optional third linker; and an optional third IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain, wherein the at least one Fc domain monomer comprises 1, 2 or 3 charge inversion amino acid mutations.

In various embodiments of the 44 aspect, the polypeptide further comprises an antibody heavy chain variable domain and a CH1 domain amino-terminal to the first IgG1 Fc domain monomer or an scFv amino-terminal to the first IgG1 Fc domain monomer. including; The first IgG1 Fc domain monomer comprises a set of two charge reversal mutations selected from those set forth in Tables 4A and 4B or a set of four charge reversal mutations selected from those set forth in Tables 4A and 4B, and a second the IgG1 Fc domain monomer comprises 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B; The first IgG1 Fc domain monomer comprises 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B, and the second IgG1 Fc domain monomer has two charges selected from those in Tables 4A and 4B. a set of inversion mutations or a set of four charge inversion mutations selected from those in Tables 4A and 4B; both the first IgG1 Fc domain monomer and the second IgG constant domain monomer comprise 1, 2 or 3 charge reversing amino acid mutations selected from Tables 4A and 4B; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein each of the first IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer is selected from Table 4A and Table 4B. , comprising two or three charge-reversing amino acid mutations; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the first IgG1 Fc domain monomer and the second IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively and wherein the third IgG1 Fc domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversions selected from those in Tables 4A and 4B. comprising a set of mutations; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the first IgG1 Fc domain monomer and the third IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively and wherein the second IgG1 domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversion mutations selected from those in Tables 4A and 4B. contains a set of; The polypeptide further comprises a third linker and a third IgG1 Fc domain monomer, wherein both the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer are 1, 2 or 3 selected from Tables 4A and 4B, respectively and wherein the first IgG1 domain monomer comprises a set of two charge inversion mutations selected from those in Tables 4A and 4B or four charge inversion mutations selected from those in Tables 4A and 4BB. contains a set of; IgGl Fc domain monomers comprising 1, 2 or 3 charge inversion amino acid mutations selected from Tables 4A and 4B have the same CH3 domain; 1, 2 or 3 charge inversion amino acid mutations selected from Tables 4A and 4B are present in the CH3 domain; The mutation is in the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); Each mutation is a single amino acid change; The mutation is in the sequence from EU numbering position G341 to EU numbering position K446 (inclusive); A mutation is a single amino acid change; The second linker and the optional third linker are GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, RGSGGS, RGSGGS, RGS, RGP, GGS, GGS, GGS, GGSG, GGGS, GGSG, GGSG, GGSG, GSGS, GSGS, GSGS, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGGGGGGG and selected from the group consisting of: the second linker and optional third linker are glycine spacers; the second linker and the optional third linker independently consist of 4-30, 4-20, 8-30, 8-20, 12-20 or 12-30 glycine residues; the second linker and optional third linker consist of 20 glycine residues; At least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position I253, wherein each amino acid mutation at EU numbering position I253 is independently 1253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I , I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y; each amino acid mutation at position 1253 is 1253A; at least one of the Fc domain monomers comprises a single amino acid mutation at EU numbering position R292; each amino acid mutation at EU numbering position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is R292P; each Fc domain monomer independently comprises or consists of an amino acid sequence selected from the group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge portion of the second Fc domain monomer and the third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the amino acid sequence EPKSCDKTHTCPPCPAPEL, and the hinge portion of the second Fc domain monomer and the third Fc domain monomer has the amino acid sequence DKTHTCPPCPAPELL; the CH2 domain of each Fc domain monomer independently comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid deletions or substitutions; the CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid substitutions; The CH2 domain of each Fc domain monomer is identical and comprises the amino acid sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid substitutions; the CH3 domain of each Fc domain monomer independently comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid substitutions; The single amino acid substitution consists of T366Y, T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R. selected from the group; each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid substitutions; up to 6 of the single amino acid substitutions are charge inversion mutations in the CH3 domain or mutations that form engineered protrusions; the single amino acid substitution is within the sequence from EU numbering position G341 to EU numbering position K447 (inclusive); The VH domain or scFv comprises the set of CDR-H1, CDR-H2 and CDR-H3 sequences shown in Table 1; The VH domain or scFv comprises CDR-H1, CDR-H2, and CDR-H3 of a VH domain comprising the sequence of an antibody set forth in Table 2; The VH domain or scFv comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of the antibody shown in Table 2, the VH sequence excluding the CDR-H1, CDR-H2, and CDR-H3 sequences is Table 2 at least 95% or 98% identical to the VH sequence of the antibody shown in The VH domain or scFv comprises the VH sequence of an antibody shown in Table 2; The VH domain or scFv comprises the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences shown in Table 1; the VH domain or scFv comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from the set of VH and VL sequences of an antibody shown in Table 2; The VH domain or scFv is a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of the antibody shown in Table 2, and the CDR-L1, CDR-L2 of the VL sequence of the antibody shown in Table 2; and a VL domain comprising CDR-L3, wherein the VH and VL domain sequences excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences are shown in Table 2 at least 95% or 98% identical to the VH and VL sequences of the antibody; The VH domain or scFv comprises the set of VH and VL sequences of an antibody shown in Table 2.

Also described are nucleic acid molecules encoding any of the aforementioned polypeptides of aspects 41, 42, 43 and 44.

Also included are expression vectors comprising nucleic acids encoding any of the polypeptides described above; a host cell containing the nucleic acid or expression vector; a host cell that further contains a nucleic acid molecule encoding a polypeptide comprising an antibody VL domain (eg, a nucleic acid molecule encoding a polypeptide comprising an antibody VL domain and an antibody CL domain); a host cell further containing a nucleic acid molecule encoding a polypeptide comprising an antibody VL domain and an antibody CL domain; a host cell further containing a nucleic acid molecule encoding a polypeptide comprising an IgG1 Fc domain monomer having no more than 10 single amino acid mutations; Host cells are described that further contain a nucleic acid molecule encoding a polypeptide comprising an IgG1 Fc domain monomer having no more than 10 single amino acid mutations. In various embodiments, the IgG1 Fc domain monomer comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45 and 47 having no more than 10, 8, 6, or 4 single amino acid mutations in the CH3 domain.

Also described are pharmaceutical compositions comprising any of the polypeptides or polypeptide complexes described herein. In various embodiments, less than 40%, 30%, 20%, 10%, 5%, 2% of the polypeptides have at least one fucose.

The polypeptides of the 41st, 42nd, 43rd and 44th aspects of the invention are useful as components of the various Fc-antigen binding domain constructs described herein. Thus, the polypeptides of any one of the first to 40 aspects, for example those that may comprise a CD38 binding domain, are the polypeptides of any one of the 41st, 42nd, 43rd and 44th aspects of the present invention. may include or consist of.

Other polypeptides useful for use in all aspects of the invention are (e.g., Y407T Y407A, F405A, T394S, T394W:Y407T, T394S:Y407A, T366W:T394S, F405T, T366S:L368A:Y407V:Y349C, S364H:F405A SEQ ID NOs: 42, 43, 45 and 47 (e.g., with no more than 8, 6, 5, 4, or 3 single amino acid substitutions) polypeptides comprising an Fc domain monomer (comprising or consisting of the amino acid sequence of any of These polypeptides may optionally comprise one, two or three charge inversion mutations of Tables 4A and 4B.

Also described herein are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain, and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second CD38 heavy chain binding domain, and

iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer;

d) a fourth polypeptide comprising a sixth Fc domain monomer;

e) a fifth polypeptide comprising a first CD38 light chain binding domain; and

f) a sixth polypeptide comprising a second CD38 light chain binding domain;

The first Fc domain monomer and the third Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the fifth Fc domain monomer together form a second Fc domain, and the fourth Fc monomer and the sixth Fc monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, wherein the second CD38 heavy chain binding domain and the second CD38 light chain bind The domains together form the second Fab.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG; Each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. including; Single amino acid substitutions are present only in the CH3 domain; The first Fc domain monomer and the third Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the first and third Fc domain monomers. amino acid substitutions; The second Fc domain monomer and the fifth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the second and fifth Fc domain monomers. amino acid substitutions, wherein the fourth Fc domain monomer and the sixth Fc domain monomer promote heterodimerization between the fourth and sixth Fc domain monomers up to 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B;

Substitutions that promote heterodimerization are selected from substitutions in Table 3.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain, and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second CD38 heavy chain binding domain, and

iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer and a first CD38 light chain binding domain; and

d) a fourth polypeptide comprising a sixth Fc domain monomer and a second CD38 light chain binding domain,

The first Fc domain monomer and the third Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the fifth Fc domain monomer together form a second Fc domain, and the fourth Fc monomer and the sixth Fc monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, wherein the second CD38 heavy chain binding domain and the second CD38 light chain bind The domains together form the second Fab.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain, and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second CD38 heavy chain binding domain, and

iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer;

d) a fourth polypeptide comprising a sixth Fc domain monomer;

e) a fifth polypeptide comprising a first CD38 light chain binding domain; and

f) a sixth polypeptide comprising a second CD38 light chain binding domain;

The first Fc domain monomer and the fifth Fc domain monomer together form a first Fc domain, the third Fc domain monomer and the sixth Fc domain monomer together form a second Fc domain, and the second Fc monomer and the fourth Fc monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, the second CD38 heavy chain binding domain and the second CD38 light chain binding The domains together form the second F.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer comprises at most 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG1; Each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. including; Single amino acid substitutions are present only in the CH3 domain; The second Fc domain monomer and the fourth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the second Fc domain monomer and the fourth Fc domain monomer. amino acid substitutions; The first Fc domain monomer and the fifth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the first and fifth Fc domain monomers. amino acid substitutions, wherein the third Fc domain monomer and the sixth Fc domain monomer promote heterodimerization between the fourth and sixth Fc domain monomers up to 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B; Substitutions that promote heterodimerization are selected from substitutions in Table 3.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a third Fc domain monomer;

iv) a first CD38 heavy chain binding domain,

v) a linker connecting the first Fc domain monomer and the second Fc domain monomer, and

vi) a first polypeptide comprising a linker connecting the second Fc domain monomer and the third Fc domain monomer;

b)

i) a fourth Fc domain monomer,

ii) a fifth Fc domain monomer,

iii) a sixth Fc domain monomer;

iv) a second CD38 heavy chain binding domain,

v) a linker connecting the fourth Fc domain monomer and the fifth Fc domain monomer, and

vi) a second polypeptide comprising a linker connecting the fifth Fc domain monomer and the sixth Fc domain monomer;

c) a third polypeptide comprising a seventh Fc domain monomer;

d) a fourth polypeptide comprising an eighth Fc domain monomer;

e) a fifth polypeptide comprising a ninth Fc domain monomer;

f) a sixth polypeptide comprising a tenth Fc domain monomer;

g) a seventh polypeptide comprising a first CD38 light chain binding domain; and

h) an eighth polypeptide comprising a second CD38 light chain binding domain;

The first Fc domain monomer and the seventh Fc domain monomer together form a first Fc domain, the fourth Fc domain monomer and the eighth Fc domain monomer together form a second Fc domain, and the second Fc monomer and the fifth Fc monomer together form a third Fc domain, the third Fc domain monomer and the ninth Fc domain monomer together form a fourth Fc domain, and the sixth Fc monomer and the tenth Fc monomer together form a fifth forming an Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, and the second CD38 heavy chain binding domain and the second CD38 light chain binding domain together form a second Fab do.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the seventh polypeptide and the eighth polypeptide are identical in sequence; the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence; the seventh polypeptide and the eighth polypeptide are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer comprises at most 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG1; the Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution, ; Single amino acid substitutions are present only in the CH3 domain; The second Fc domain monomer and the fifth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the second Fc domain monomer and the fifth Fc domain monomer. amino acid substitutions; The first Fc domain monomer and the seventh Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the first and seventh Fc domain monomers. amino acid substitutions, wherein the fourth Fc domain monomer and the eighth Fc domain monomer promote heterodimerization between the fourth and eighth Fc domain monomers up to 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution, wherein the third Fc domain monomer and the ninth Fc domain monomer promote heterodimerization between the third Fc domain monomer and the ninth Fc domain monomer up to 8, 7, 6 , 5, 4, 3, 2 or 1 single amino acid substitution, wherein the sixth Fc domain monomer and the tenth Fc domain monomer promote heterodimerization between the sixth Fc domain monomer and the tenth Fc domain monomer. up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B; Substitutions that promote heterodimerization are selected from substitutions in Table 3.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a third Fc domain monomer;

iv) a first CD38 heavy chain binding domain,

v) a linker connecting the first Fc domain monomer and the second Fc domain monomer, and

vi) a first polypeptide comprising a linker connecting the second Fc domain monomer and the third Fc domain monomer;

b)

i) a fourth Fc domain monomer,

ii) a fifth Fc domain monomer,

iii) a sixth Fc domain monomer;

iv) a second CD38 heavy chain binding domain,

v) a linker connecting the fourth Fc domain monomer and the fifth Fc domain monomer, and

vi) a second polypeptide comprising a linker connecting the fifth Fc domain monomer and the sixth Fc domain monomer;

c) a third polypeptide comprising a seventh Fc domain monomer;

d) a fourth polypeptide comprising an eighth Fc domain monomer;

e) a fifth polypeptide comprising a ninth Fc domain monomer and a first CD38 light chain binding domain; and

f) a sixth polypeptide comprising a tenth Fc domain monomer and a second CD38 light chain binding domain,

The first Fc domain monomer and the seventh Fc domain monomer together form a first Fc domain, the fourth Fc domain monomer and the eighth Fc domain monomer together form a second Fc domain, and the second Fc monomer and the fifth Fc monomer together form a third Fc domain, the third Fc domain monomer and the ninth Fc domain monomer together form a fourth Fc domain, and the sixth Fc monomer and the tenth Fc monomer together form a fifth forming an Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, and the second CD38 heavy chain binding domain and the second CD38 light chain binding domain together form a second Fab do.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a third Fc domain monomer;

iv) a first CD38 heavy chain binding domain,

v) a linker connecting the first Fc domain monomer and the second Fc domain monomer, and

vi) a first polypeptide comprising a linker connecting the second Fc domain monomer and the third Fc domain monomer;

b)

i) a fourth Fc domain monomer,

ii) a fifth Fc domain monomer,

iii) a sixth Fc domain monomer;

iv) a second CD38 heavy chain binding domain,

v) a linker connecting the fourth Fc domain monomer and the fifth Fc domain monomer, and

vi) a second polypeptide comprising a linker connecting the fifth Fc domain monomer and the sixth Fc domain monomer;

c) a third polypeptide comprising a seventh Fc domain monomer;

d) a fourth polypeptide comprising an eighth Fc domain monomer;

e) a fifth polypeptide comprising a ninth Fc domain monomer;

f) a sixth polypeptide comprising a tenth Fc domain monomer;

g) a seventh polypeptide comprising a first CD38 light chain binding domain; and

h) an eighth polypeptide comprising a second CD38 light chain binding domain;

The first Fc domain monomer and the fourth Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the seventh Fc domain monomer together form a second Fc domain, and the fifth Fc monomer and the eighth Fc monomer together form a third Fc domain, the third Fc domain monomer and the ninth Fc domain monomer together form a fourth Fc domain, and the sixth Fc monomer and the tenth Fc monomer together form a fifth forming an Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, and the second CD38 heavy chain binding domain and the second CD38 light chain binding domain together form a second Fab do.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the seventh polypeptide and the eighth polypeptide are identical in sequence; the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence; the seventh polypeptide and the eighth polypeptide are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer comprises at most 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG1; Each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. including; Single amino acid substitutions are present only in the CH3 domain; The first Fc domain monomer and the fourth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the first and fourth Fc domain monomers. amino acid substitutions; The second Fc domain monomer and the seventh Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the second Fc domain monomer and the seventh Fc domain monomer. amino acid substitutions, wherein the fifth Fc domain monomer and the eighth Fc domain monomer promote heterodimerization between the fifth Fc domain monomer and the eighth Fc domain monomer at most 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution, wherein the third Fc domain monomer and the ninth Fc domain monomer promote heterodimerization between the third Fc domain monomer and the ninth Fc domain monomer up to 8, 7, 6 , 5, 4, 3, 2 or 1 single amino acid substitution, wherein the sixth Fc domain monomer and the tenth Fc domain monomer promote heterodimerization between the sixth Fc domain monomer and the tenth Fc domain monomer. up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B; Substitutions that promote heterodimerization are selected from substitutions in Table 3.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a third Fc domain monomer;

iv) a first CD38 heavy chain binding domain,

v) a linker connecting the first Fc domain monomer and the second Fc domain monomer, and

vi) a first polypeptide comprising a linker connecting the second Fc domain monomer and the third Fc domain monomer;

b)

i) a fourth Fc domain monomer,

ii) a fifth Fc domain monomer,

iii) a sixth Fc domain monomer;

iv) a second CD38 heavy chain binding domain,

v) a linker connecting the fourth Fc domain monomer and the fifth Fc domain monomer, and

vi) a second polypeptide comprising a linker connecting the fifth Fc domain monomer and the sixth Fc domain monomer;

c) a third polypeptide comprising a seventh Fc domain monomer;

d) a fourth polypeptide comprising an eighth Fc domain monomer;

e) a fifth polypeptide comprising a ninth Fc domain monomer and a first CD38 light chain binding domain;

f) a sixth polypeptide comprising a tenth Fc domain monomer and a second CD38 light chain binding domain,

The first Fc domain monomer and the fourth Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the seventh Fc domain monomer together form a second Fc domain, and the fifth Fc monomer and the eighth Fc monomer together form a third Fc domain, the third Fc domain monomer and the ninth Fc domain monomer together form a fourth Fc domain, and the sixth Fc monomer and the tenth Fc monomer together form a fifth forming an Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, and the second CD38 heavy chain binding domain and the second CD38 light chain binding domain together form a second Fab do.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer; and

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer and a first CD38 heavy chain binding domain; and

d) a fourth polypeptide comprising a sixth Fc domain monomer and a second CD38 heavy chain binding domain;

e) a fifth polypeptide comprising a first CD38 light chain binding domain; and

f) a sixth polypeptide comprising a second CD38 light chain binding domain;

The first Fc domain monomer and the fifth Fc domain monomer together form a first Fc domain, the third Fc domain monomer and the sixth Fc domain monomer together form a second Fc domain, and the second Fc domain monomer and the fourth Fc domain monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, the second CD38 heavy chain binding domain and the second CD38 The light chain binding domains together form the second Fab.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer comprises at most 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG1; Each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. including; Single amino acid substitutions are only present in the CH3 domain; The second Fc domain monomer and the fourth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the second Fc domain monomer and the fourth Fc domain monomer. amino acid substitutions; The first Fc domain monomer and the fifth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the first and fifth Fc domain monomers. amino acid substitutions, wherein the third Fc domain monomer and the sixth Fc domain monomer promote heterodimerization between the third and sixth Fc domain monomers up to 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B; Substitutions that promote heterodimerization are selected from substitutions in Table 3.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain, and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second CD38 heavy chain binding domain, and

iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer and a third CD38 heavy chain binding domain;

d) a fourth polypeptide comprising a sixth Fc domain monomer and a fourth CD38 light chain binding domain;

e) a fifth polypeptide comprising a first CD38 light chain binding domain;

f) a sixth polypeptide comprising a second CD38 light chain binding domain;

g) a seventh polypeptide comprising a third CD38 light chain binding domain; and

h) an eighth polypeptide comprising a fourth CD38 light chain binding domain;

The first Fc domain monomer and the fifth Fc domain monomer together form a first Fc domain, the third Fc domain monomer and the sixth Fc domain monomer together form a second Fc domain, and the second Fc monomer and a fourth Fc monomer together form a third Fc domain, said first CD38 light chain binding domain and third CD38 heavy chain binding domain together form a first Fab, said second CD38 light chain binding domain and a fourth CD38 heavy chain binding domain the domains together form a second Fab, the third CD38 light chain binding domain and the first CD38 heavy chain binding domain together form a third Fab, and the fourth CD38 light chain binding domain and the second CD38 heavy chain binding domain together form a first 2 Fab is formed.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth, sixth, seventh, and eighth polypeptides are identical in sequence; the first polypeptide and the second polypeptide are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth, sixth, seventh, and eighth polypeptides are identical in sequence; the CH3 domain of each Fc domain monomer contains up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution; the CH3 domain of each Fc domain monomer comprises at most 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution relative to the amino acid sequence of human IgG1; Each Fc domain monomer independently comprises the amino acid sequence of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. including; Single amino acid substitutions are only present in the CH3 domain; The second Fc domain monomer and the fourth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes homodimerization between the second Fc domain monomer and the fourth Fc domain monomer. amino acid substitutions; The first Fc domain monomer and the fifth Fc domain monomer may contain up to 8, 7, 6, 5, 4, 3, 2 or 1 single unit that promotes heterodimerization between the first and fifth Fc domain monomers. amino acid substitutions, wherein the third Fc domain monomer and the sixth Fc domain monomer promote heterodimerization between the third and sixth Fc domain monomers up to 8, 7, 6, 5, 4, 3 , 2 or 1 single amino acid substitution; substitutions that promote homodimerization are selected from substitutions in Tables 4A and 4B; Substitutions that promote heterodimerization are selected from substitutions in Table 3.

다양한 실시 형태에서, 각각의 링커는 GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG , AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG, GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; at least one of the Fc domain monomers comprises a substitution at EU position 1253; Each amino acid substitution at EU position I253 is, independently, I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V , and 1253Y; at least one of the Fc domain monomers comprises a substitution at EU position R292; each amino acid substitution at EU position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y; At least one of the Fc domain monomers is T366Y, T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K, D399R, E357K, E357R, D356K, and a substitution selected from the group consisting of D356R; The hinge of each Fc domain monomer independently comprises or consists of an amino acid sequence selected from the group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL.

In all aspects of the invention, some or all of the Fc domain monomers (e.g. (e.g. only in the CH3 domain) SEQ ID NOs: 42, 43 having no more than 10, 8, 6 or 4 single amino acid substitutions , 45 and 47) may have one or both of the E345K and E430G amino acid substitutions, in addition to other amino acid substitutions or modifications. E345K and E430G amino acid substitutions may increase Fc domain multimerization.

Also described are Fc-antigen binding domain constructs, said Fc-antigen binding domain constructs comprising

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain (eg, comprising a VH domain and a CH1 domain), and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b)

i) a third Fc domain monomer,

ii) a fourth Fc domain monomer,

iii) a second CD38 heavy chain binding domain (eg, comprising a VH domain and a CH1 domain), and

iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;

c) a third polypeptide comprising a fifth Fc domain monomer;

d) a fourth polypeptide comprising a sixth Fc domain monomer;

e) a fifth polypeptide comprising a first CD38 light chain binding domain (eg, comprising a VL domain and a CL domain); and

f) a sixth polypeptide comprising a second CD38 light chain binding domain (eg, comprising a VL domain and a CL domain);

The first Fc domain monomer and the third Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the fifth Fc domain monomer together form a second Fc domain, and the fourth Fc monomer and the sixth Fc monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab, wherein the second CD38 heavy chain binding domain and the second CD38 light chain bind The domains together form the second Fab.

In various embodiments, the first and second polypeptides are identical in sequence; the third and fourth polypeptides are identical in sequence; the fifth and sixth polypeptides are identical in sequence; the first and second polypeptides are at least 95% identical to SEQ ID NO: B, the third and fourth polypeptides are at least 95% identical to SEQ ID NO: C, and the fifth and sixth polypeptides are at least 95% identical to SEQ ID NO: A % equal; the first and second polypeptides are at least 98% identical to SEQ ID NO: B, the third and fourth polypeptides are at least 98% identical to SEQ ID NO: C, and the fifth and sixth polypeptides are at least 98% identical to SEQ ID NO: A % equal; the first and second polypeptides comprise or consist of SEQ ID NO: B, the third and fourth polypeptides comprise or consist of SEQ ID NO: C, and the fifth and sixth polypeptides comprise or consist of SEQ ID NO: A This is done; The first and second polypeptides have the same sequence, the third and fourth polypeptides have the same sequence, and the fifth and sixth polypeptides have the same sequence.

Also disclosed is a composition, said composition comprising:

a)

i) a first Fc domain monomer,

ii) a second Fc domain monomer,

iii) a first CD38 heavy chain binding domain, and

iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;

b) a second polypeptide comprising a third Fc domain monomer; and

c) a third polypeptide comprising a CD38 light chain binding domain.

In various embodiments, the first polypeptide is at least 95% identical to SEQ ID NO: B, the second polypeptide is at least 95% identical to SEQ ID NO: C, and the third polypeptide is at least 95% identical to SEQ ID NO: A; the first polypeptide is at least 98% identical to SEQ ID NO: B, the second polypeptide is at least 98% identical to SEQ ID NO: C, and the third polypeptide is at least 98% identical to SEQ ID NO: A; The first polypeptide comprises or consists of SEQ ID NO: B, the second polypeptide comprises or consists of SEQ ID NO: C, and the third polypeptide comprises or consists of SEQ ID NO: A.

Justice:

As used herein, the term “Fc domain monomer” refers to at least a hinge domain and second and third antibody constant domains (C _H 2 and C _H 3 ) or functional fragments thereof (eg, at least a hinge domain or functional fragments thereof, CH2 domains or functional fragments thereof, and CH3 domains or functional fragments thereof) (eg, (i) capable of dimerizing with other Fc domain monomers to form an Fc domain, and (ii) binding to an Fc receptor) fragments capable of). Preferred Fc domain monomers comprise, from amino-terminus to carboxy-terminus, at least a portion of an IgG1 hinge, an IgG1 CH2 domain and an IgG1 CH3 domain. Thus, an Fc domain monomer, e.g., a human IgG1 Fc domain monomer, can be from E316 to G446 or K447, from P317 to G446 or K447, from K318 to G446 or K447, from K318 to G446 or K447, from S319 to G446 or K447, C320 to G446 or K447, D321 to G446 or K447, K322 to G446 or K447, T323 to G446 or K447, K323 to G446 or K447, H324 to G446 or K447, T325 to G446 or K447, or It can be extended from C326 to G446 or K447. The Fc domain monomer may be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (eg, IgG). Additionally, the Fc domain monomer may be of an IgG subtype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4) (eg, human IgG1). Human IgG1 Fc domain monomers are used in the examples described herein. The complete hinge domain of human IgG1 extends from EU numbering E316 to P230 or L235, the CH2 domain extends from A231 or G236 to K340, and the CH3 domain extends from G341 to K447. There are different views on the position of the last amino acid of the hinge domain. It is either P230 or L235. In many examples herein, the CH3 domain does not comprise K347. Thus, the CH3 domain may be G341 to G446. In many examples herein, the hinge domain may comprise E216-L235. This is the case, for example, when the hinge is carboxy-terminal to the CH1 domain or the CD38 binding domain. In some cases, for example, when the hinge is at the amino-terminus of the polypeptide, Asp at EU numbering 221 is mutated to Gin. The Fc domain monomer does not contain any portion of an immunoglobulin that can act as an antigen-recognition region, for example it does not contain a variable domain or a complementarity determining region (CDR). Fc domain monomers vary from wild-type (eg human) Fc domain monomer sequences to as many as 10 (eg, 1-10, 1-8) altering the interaction between the Fc domain and the Fc receptor. , 1 to 6, 1 to 4 amino acid substitutions, additions, or deletions). Fc domain monomers may vary from the wild-type Fc domain monomer sequence to as many as ten (e.g., single amino acid changes) (e.g., 1 to 10, 1 to 8) altering interactions between the Fc domain monomers. , 1 to 6, 1 to 4 amino acid substitutions, additions, or deletions). In certain embodiments, there are at most 10, 8, 6 or 5 single amino acid substitutions on the CH3 domain relative to the human IgG1 CH3 domain sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. Examples of suitable variations are known in the art.

As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers capable of binding an Fc receptor. In the wild-type Fc domain, the two Fc domain monomers are not only responsible for one or more disulfide bonds formed between the hinge domains of the two dimerizable Fc domain monomers, as well as by interaction between the two C _H 3 antibody constant domains. dimerized by

As used herein, the term “Fc-antigen binding domain construct” refers to an associated polypeptide chain comprising at least one “antigen binding domain” and forming at least two Fc domains as described herein. The Fc-antigen binding domain constructs described herein may comprise Fc domain monomers having the same or different sequences. For example, an Fc-antigen binding domain construct may have three Fc domains, two comprising an IgG1 or IgG1-derived Fc domain monomer and a third comprising an IgG2 or IgG2-derived Fc domain monomer. include In another example, an Fc-antigen binding domain construct may have three Fc domains, two of which comprise a "protrusion pair into a cavity" and a third not comprising a "protrusion pair into a cavity" does not The Fc domain forms a minimal structure that binds to an Fc receptor, for example FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, or FcγRIV.

As used herein, the term “antigen binding domain” refers to a set of peptides, polypeptides, or associated polypeptides capable of specifically binding to a target molecule. In some embodiments, an “antigen binding domain” is the minimal sequence of an antibody that binds with specificity for the antigen bound by the antibody. Surface plasmon resonance (SPR) or various immunoassays known in the art, such as Western Blot or ELISA, can be used to assess antibody specificity for an antigen. In some embodiments, an “antigen binding domain” is a variable domain or complementarity determining region (CDR) of an antibody, e.g., one or more CDRs of an antibody set forth in Table 1, one or more CDRs of an antibody set forth in Table 2, or VH and/or VL domains of a given antibody. In some embodiments, the CD38 binding domain may comprise a VH domain and a CH1 domain, optionally along with a VL domain. In another embodiment, the antigen (eg, CD38) binding domain is a Fab fragment or scFv of an antibody. Thus, a CD38 binding domain may comprise a “CD38 heavy chain binding domain” comprising or consisting of a VH domain and a CH1 domain and a “CD38 light chain binding domain” comprising or consisting of a _VL domain and a CL domain. The CD38 binding domain can also be a synthetically engineered peptide that specifically binds a target, such as a fibronectin-based binding protein (eg, a fibronectin type III domain (FN3) monobody).

As used herein, the term "complementarity determining region" (CDR) refers to the amino acid residues of an antibody variable domain, wherein their presence is required for CD38 binding. Each variable domain typically has three CDR regions identified as CDR-L1, CDR-L2 and CDR-L3, and CDR-H1, CDR-H2, and CDR-H3. Each complementarity determining region consists of amino acid residues from a "complementarity determining region" as defined by Kabat (i.e., approximately, residues 24-34 (CDR-L1), 50-56 in the light chain variable domain) CDR-L2), and 89-97 (CDR-L3), and residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in the heavy chain variable domain; [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]) and/or amino acid residues from “hypervariable loops” (i.e., approximately, residues 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3) in the light chain variable domain and residues 26-32 (CDR-) in the heavy chain variable domain H1), 53-55 (CDR-H2), and 96-101 (CDR-H3); Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some cases, the complementarity determining regions may include amino acids from both the CDR regions defined according to Kabat and hypervariable loops.

"Framework regions" (hereinafter FRs) are variable domain residues other than CDR residues. Each variable domain has four FRs, typically identified as FR1, FR2, FR3 and FR4. When the CDRs are defined according to Kabat, the light chain FR residues are located approximately at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues The residues are located approximately at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) within the heavy chain residues. When the CDR comprises amino acid residues from a hypervariable loop, the light chain FR residues are approximately residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain. ), and the heavy chain FR residues are located approximately at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) within the heavy chain residues. In some cases, if a CDR comprises amino acids from both the CDR as defined by Kabat and that of the hypervariable loop, the FR residues will be adjusted accordingly.

An “Fv” fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which may be covalent in nature, for example in the form of an scFv. In this conformation, the three CDRs of each variable domain interact to define a CD38 binding site on the surface of the V _H -V _L dimer.

A “Fab” fragment contains a variable domain and a constant domain of a light chain, and a variable domain and a first constant domain of a heavy chain ( _CH 1 ). F(ab′) ₂ antibody fragments comprise a pair of Fab fragments that are generally covalently linked by hinge cysteines near the carboxy-terminus.

A “single chain Fv” or “scFv” antibody fragment comprises the V _H and V _L domains of an antibody within a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the V _H domain and the V _L domain, which polypeptide linker enables the scFv to form the desired structure for CD38 binding.

As used herein, the term “antibody constant domain” refers to the constant region domain of an antibody (eg, a C _L antibody constant domain, a C _H 1 antibody constant domain, a C _H 2 antibody constant domain, or a C _H 3 antibody). constant domain).

As used herein, the term “promoting” refers to, e.g., promotes and favors the formation of an Fc domain from two Fc domain monomers that have a higher binding affinity to each other than other, separate Fc domain monomers. For example, it means to favor its formation. As described herein, the two Fc domain monomers that combine to form an Fc domain contain compatible amino acid modifications at the interface of their respective C _H 3 antibody constant domains (eg, engineered protrusions and engineered cavities, and/or or an electrostatic steering mutation). A compatible amino acid modification facilitates or favors the selective interaction of such Fc domain monomers with each other over selective interaction with other Fc domain monomers that lack or have incompatible amino acid modifications. This is because, due to amino acid modifications at the interface of the two interacting C _H 3 antibody constant domains, these Fc domain monomers have a higher affinity for each other than for other Fc domain monomers lacking amino acid modifications. happens

As used herein, the term “dimerization selectivity module” refers to a sequence of Fc domain monomers that promotes favorable pairing between two Fc domain monomers. A “complementary” dimerization selectivity module is a dimerization selectivity module that promotes or favors the selective interaction of two Fc domain monomers with each other. Complementary dimerization selectivity modules may have identical or different sequences. Exemplary complementary dimerization selectivity modules are described herein.

As used herein, the term "engineered cavity" refers to substituting at least one of the original amino acid residues in the C _H 3 antibody constant domain with a different amino acid residue having a smaller side chain volume than the original amino acid residue, whereby refers to the creation of a three-dimensional cavity within the C _H 3 antibody constant domain. The term “native amino acid residue” refers to a naturally occurring amino acid residue encoded by the genetic code of a wild-type C _H 3 antibody constant domain.

As used herein, the term “engineered protrusion” refers to substituting at least one of the original amino acid residues in the C _H 3 antibody constant domain with a different amino acid residue having a larger side chain volume than the original amino acid residue, whereby C _H 3 Refers to the creation of three-dimensional projections within the antibody constant domain. The term “native amino acid residue” refers to a naturally occurring amino acid residue encoded by the genetic code of a wild-type C _H 3 antibody constant domain.

As used herein, the term “pair of projections into a cavity” describes an Fc domain comprising two Fc domain monomers, wherein the first Fc domain monomer defines an engineered cavity within its C _H 3 antibody constant domain. whereas the second Fc domain monomer comprises engineered protrusions within its C _H 3 antibody constant domain. In the pair of protrusions into the cavity, the engineered protrusions in the C _H 3 antibody constant domain of the first Fc domain monomer do not significantly perturb the normal association of the dimers at the C _H 3 antibody constant domain interface, the second Fc positioned to interact with the engineered cavity of the C _H 3 antibody constant domain of the domain monomer.

As used herein, the term “heterodimeric Fc domain” refers to an Fc domain formed by heterodimerization of two Fc domain monomers, wherein the two Fc domain monomers are those two Fc domains. It contains different charge reversal mutations that promote the favorable formation of monomers (see, eg, mutations in Tables 4A and 4B). In an Fc construct with three Fc domains - one carboxyl-terminal "stem" Fc domain and two amino-terminal "branched" Fc domains - each amino-terminal "branched" Fc domain is heterodimeric Fc domain (also called "branched heterodimeric Fc domain").

As used herein, the term "structurally identical" in reference to a population of Fc-antigen binding domain constructs refers to constructs that are assemblies of identical polypeptide sequences in identical ratios and conformation, and any post-translational modifications ( eg, glycosylation).

As used herein, the term “homodimeric Fc domain” refers to an Fc domain formed by homodimerization of two Fc domain monomers, wherein the two Fc domain monomers have identical charge inversion mutations ( See, for example, mutations in Tables 5 and 6). In an Fc construct with three Fc domains - one carboxyl-terminal "stem" Fc domain and two amino-terminal "branch" Fc domains - the carboxy-terminal "stem" Fc domain is a homodimeric Fc domain (also called "stem homodimeric Fc domain").

As used herein, the term “heterodimerization selectivity module” refers to C _H 3 of Fc domain monomers to promote favorable heterodimerization of two Fc domain monomers with compatible heterodimerization selectivity modules. refers to engineered protrusions, engineered cavities, and certain charge inversion amino acid substitutions that can be made in an antibody constant domain. Fc domain monomers containing a heterodimerization selectivity module can bind to form a heterodimeric Fc domain. Examples of heterodimerization selectivity modules are shown in Tables 3 and 4.

As used herein, the term “homodimerization selectivity module” refers to charged residues at the interface between C _H 3 domains that promote homodimerization of Fc domain monomers to form homodimeric Fc domains. refers to a charge reversal mutation in the Fc domain monomer at at least two positions within the ring of Examples of homodimerization selectivity modules are shown in Tables 4 and 5.

As used herein, the term “linked” refers to two or more elements, components, or Used to describe the association or attachment of protein domains, eg, polypeptides. For example, two single polypeptides can be linked to form one continuous protein structure via chemical conjugation, chemical bonding, peptide linkers, or any other means of covalent bonding. In some embodiments, the CD38 binding domain is linked to an Fc domain monomer by being expressed from a contiguous nucleic acid sequence encoding both the CD38 binding domain and the Fc domain monomer. In another embodiment, the CD38 binding domain is linked to the Fc domain monomer by a peptide linker, wherein the N-terminus of the peptide linker is linked to the C-terminus of the CD38 binding domain via a chemical bond, e.g., a peptide bond; The C-terminus of the linker is connected to the N-terminus of the Fc domain monomer via a chemical bond, for example a peptide bond.

As used herein, the term “associated” means that the polypeptides (or sequences within one single polypeptide) are described herein in an Fc-antigen binding domain construct (eg, an Fc having three Fc domains). - used to describe interactions, such as hydrogen bonding, hydrophobic interactions, or ionic interactions, between polypeptides (or sequences within one single polypeptide) that allow them to be positioned to form an antigen binding domain construct). do. For example, in some embodiments, four polypeptides, e.g., two polypeptides each comprising two Fc domain monomers and two polypeptides each comprising one Fc domain monomer, associate to form three Fc domains. Fc constructs with (eg, as shown in FIGS. 50 and 51 ). The four polypeptides can associate via their respective Fc domain monomers. Association between polypeptides does not involve covalent interactions.

As used herein, the term “linker” refers to a linkage between two elements, eg, protein domains. The linker may be a covalent bond or a spacer. The term “bond” refers to a chemical bond, such as an amide bond or a disulfide bond, or any kind of bond resulting from a chemical reaction, such as a chemical bond. The term “spacer” refers to a moiety (eg, a polyethylene glycol (PEG) polymer) that exists between two polypeptides or polypeptide domains to provide space and/or flexibility between the two polypeptides or polypeptide domains or amino acid sequence (eg, 3 to 200 amino acids, 3 to 150 amino acids, or 3 to 100 amino acids sequence). An amino acid spacer is part of the primary sequence of a polypeptide (eg, linked to spaced apart polypeptides or polypeptide domains via a polypeptide backbone). For example, the formation of a disulfide bond between two Fc domain monomers or two hinge regions forming an Fc domain is not considered a linker.

As used herein, the term “glycine spacer” refers to a linker containing only glycine that connects two Fc domain monomers in tandem in tandem. The glycine spacer may contain at least 4, 8, or 12 glycines (eg, 4 to 30, 8 to 30, or 12 to 30 glycines; e.g., 12 to 30, 4, 5, 6, 7) , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycines) may contain. In some embodiments, the glycine spacer has the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).

As used herein, the term “albumin-binding peptide” refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and the function of binding to serum albumin. Albumin-binding peptides can be of different origins, for example from human, mouse, or rat. In some embodiments of the invention, an albumin-binding peptide is fused to the C-terminus of an Fc domain monomer to increase the serum half-life of the Fc-antigen binding domain construct. The albumin-binding peptide may be fused to the N-terminus or C-terminus of the Fc domain monomer, either directly or via a linker.

As used herein, the term “purified peptide” refers to a peptide of any length that can be used in the purification, isolation, or identification of a polypeptide. Purifying peptides can be linked to polypeptides that aid in, for example, isolating the polypeptide and/or purifying the polypeptide from a cell lysate mixture. In some embodiments, the purified peptide binds to another moiety with specific affinity for the purified peptide. In some embodiments, those moieties that specifically bind the purified peptide are attached to a solid support, such as a matrix, resin, or agarose bead. Examples of purified peptides that can be linked to Fc-antigen binding domain constructs are described in further detail herein.

As used herein, the term “multimer” refers to a molecule comprising at least two associated Fc constructs or Fc-antigen binding domain constructs described herein.

As used herein, the term “polynucleotide” refers to oligonucleotides, or nucleotides, and fragments or portions thereof, and genomic or synthetic origin, which may be single-stranded or double-stranded and may represent either the sense or anti-sense strand. refers to the DNA or RNA of A single polynucleotide is translated into a single polypeptide.

As used herein, the term “polypeptide” describes a homopolymer in which the monomers are amino acid residues linked together via amide bonds. A polypeptide is intended to include any amino acid sequence that occurs naturally, recombinantly, or synthetically.

As used herein, the term “amino acid position” refers to the position number of an amino acid within a protein or protein domain. Amino acid positions are numbered using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., ed 5, 1991), (e.g., CDR and FR regions). for), otherwise EU numbering is used.

24A-24D show human IgG1 Fc domains numbered using the EU numbering system.

As used herein, the term "amino acid modification" refers to the pharmacokinetics (PK) and/or the Fc construct as compared to a reference sequence (eg, wild-type, unmutated, or unmodified Fc sequence). or pharmacodynamic (PD) properties, serum half-life, effector function (e.g., cell lysis (e.g., antibody-dependent cell-mediated toxicity (ADCC) and/or complement dependent cytotoxic activity (CDC)), phagocytosis (eg, antibody dependent cellular phagocytosis (ADCP) and/or complement-dependent cellular cytotoxicity (CDCC)), immune activation, and T-cell activation), Fc receptors (eg, Fc-gamma receptors) (FcγR) (e.g., FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), and/or FcγRIIIb (CD16b)), Fc-alpha receptor (FcαR), Fc-epsilon receptor ( FcεR), and/or the neonatal Fc receptor (FcRn)), affinity for proteins involved in the complement cascade (eg Clq), post-translational modifications (eg glycosylation, sialylation) , aggregation properties (eg, the ability to form dimers (eg, homodimers and/or heterodimers) and/or multimers), and biophysical properties (eg, with _CH 1 and properties that alter the interaction between C _L and/or alter stability and/or alter sensitivity to temperature and/or pH) Refers to alterations in Fc domain polypeptide sequence that may promote improved efficacy. Amino acid modifications include amino acid substitutions, deletions, and/or insertions. In some embodiments, the amino acid modification is a modification of a single amino acid. In other embodiments, the amino acid modification is a modification of multiple (eg, more than one) amino acids. Amino acid modifications may include combinations of amino acid substitutions, deletions, and/or insertions. Descriptions of amino acid modifications include genetic (i.e., DNA and RNA) alterations, such as point mutations (e.g., exchange of a single nucleotide with another nucleotide), insertions and deletions of the nucleotide sequence encoding the Fc polypeptide (e.g., addition and/or removal of one or more nucleotides).

In certain embodiments, at least one (eg, 1, 2, or 3) Fc domain monomers in the Fc construct or Fc-antigen binding domain construct comprise amino acid modifications (eg, substitutions). In some cases, the at least one Fc domain monomer has one or more (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20) amino acid modifications (e.g., substitutions ) is included.

As used herein, the term "percent (%) identity" refers to a candidate sequence, e.g., an Fc described herein, that is identical to amino acid (or nucleic acid) residues of a reference sequence, e.g., a sequence of a wild-type Fc domain monomer. - refers to the percentage of amino acid (or nucleic acid) residues of the sequence of the Fc domain monomer in the antigen binding domain construct, which is obtained after aligning the sequences and introducing gaps if necessary to achieve maximum percent identity (i.e., Gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and heterologous sequences may be discarded for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the % amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or relative to a given reference sequence (which is alternatively a given % amino acid (or nucleic acid) sequence identity (which may be referred to as a given candidate sequence having or comprising sequence identity) is calculated as follows:

100 x (fraction of A/B)

(where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence). In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the % amino acid (or nucleic acid) sequence identity of the candidate sequence with the reference sequence is equal to the % amino acid (or nucleic acid) sequence identity of the reference sequence with the candidate sequence. It won't be the same.

In certain embodiments, a reference sequence aligned for comparison with a candidate sequence has 50% to 100% identity (e.g., 50% to 100% identity (e.g., For example, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 92% to 100%, 95% to 100%, 97% to 100%, 99% to 100%, or 99.5% to 100% identity). The length of an aligned candidate sequence for comparison purposes is at least 30%, such as at least 40%, such as at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

In some embodiments, the Fc domain monomers in an Fc construct described herein (eg, an Fc-antigen binding domain construct having three Fc domains) are those of a wild-type Fc domain monomer (eg, SEQ ID NO: 42). a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence. In some embodiments, the Fc domain monomer in an Fc construct described herein (eg, an Fc-antigen binding domain construct having three Fc domains) is any of SEQ ID NOs: 44, 46, 48, and 50-53. a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to one sequence. In certain embodiments, the Fc domain monomers in the Fc construct may have a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequences of SEQ ID NOs: 48, 52, and 53.

In some embodiments, the spacer between the two Fc domain monomers is at least 75% with the sequence of any one of SEQ ID NOs: 1-36 (e.g., SEQ ID NOs: 17, 18, 26, and 27) further described herein. equal (at least 75%, 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, 99.5%, or 100% identical) may have a sequence.

As used herein, the term “host cell” refers to a vehicle that contains the necessary cellular components, eg, organelles, required to express a protein from the corresponding nucleic acid. Nucleic acids are typically contained in nucleic acid vectors that can be introduced into host cells by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (eg, a CHO cell). As described herein, a host cell is used to express one or more polypeptides encoding the desired domains, which may then be combined to form the desired Fc-antigen binding domain construct.

As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical formulation containing the active ingredient as well as one or more excipients and diluents that render the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable component compatible with the Fc-antigen binding domain construct. Pharmaceutical compositions are typically in aqueous form for intravenous or subcutaneous administration.

As used herein, a "substantially homogeneous population" of a polypeptide or Fc construct is defined as at least 50% of the polypeptide or Fc construct in the composition (eg, cell culture medium or pharmaceutical composition) is non-reducing. They have the same number of Fc domains as determined by SDS gel electrophoresis or size exclusion chromatography. A substantially homogeneous population of polypeptides or Fc constructs can be obtained prior to purification, or after protein A or protein G purification, or only after any Fab or Fc-specific affinity chromatography. In various embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the polypeptides or Fc constructs in the composition have the same number of Fc domains. In other embodiments, up to 85%, 90%, 92%, or 95% of the polypeptides or Fc constructs in the composition have the same number of Fc domains.

As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, a pharmaceutically acceptable carrier should provide adequate pharmaceutical stability to the Fc-antigen binding domain construct. The nature of the carrier differs depending on the mode of administration. For example, for oral administration, solid carriers are preferred; For intravenous administration, aqueous carriers (eg, WFI, and/or buffered solutions) are commonly used.

As used herein, "therapeutically effective amount" refers to an amount, e.g., a pharmaceutical dose, effective to induce a desired biological effect in a subject or patient or to treat a patient having a disease or disorder described herein. . It should also be understood herein that a "pharmaceutically effective amount" may be construed as an amount that provides the desired therapeutic effect, taken alone or in combination with other therapeutic agents, either in a single dose or by any dosage or route. do.

As used herein, the terms 'fragment' and the term 'part' may be used interchangeably.

1 is an illustration of an Fc-antigen binding domain construct (Construct 1) containing two Fc domains and a CD38 binding domain. Each Fc domain is a dimer of two Fc domain monomers. Two of the Fc domain monomers (106, 108) contain projections within their C _H 3 antibody constant domains, while the other two Fc domain monomers (112, 114) are juxtaposed within their C _H 3 antibody constant domains. contain cavities in place. This construct is formed from a polypeptide containing three Fc domain monomers. The first polypeptide (102) contains two projection-containing Fc domain monomers (106, 108) linked by a spacer in tandem in tandem to a CD38 binding domain (110) containing a V _H domain on the N-terminus. The V _L containing domain 104 is linked to the V _H domain. Each of the second and third polypeptides 112 , 114 contains a co-containing Fc domain monomer.
2 is an illustration of an Fc-antigen binding domain construct (Construct 2) containing three Fc domains and a CD38 binding domain. This construct is formed from a polypeptide containing four Fc domain monomers. The first polypeptide 202 contains three protrusion-containing Fc domains 206, 208, 210 connected by spacers in tandem in tandem to a CD38 binding domain 212 containing a V _H domain on the N-terminus. . The V _L containing domain 204 is linked to the V _H domain. Each of the second, third, and fourth polypeptides 214 , 216 , 218 contains a co-containing Fc domain monomer.
3 is an illustration of an Fc-antigen binding domain construct (Construct 3) containing two Fc domains and two CD38 binding domains. This construct is formed from a polypeptide containing three Fc domain monomers. The first polypeptide (302) contains two projection-containing Fc domain monomers (304, 306) linked by a spacer in tandem in tandem. Each of the second and third polypeptides 320, 322 contains co-containing Fc domain monomers 310, 314 linked in tandem to a CD38 binding domain 316, 318 containing a V _H domain on the N-terminus. do. V _L containing domains 308 , 312 are linked to each V _H domain.
4 is an illustration of an Fc-antigen binding domain construct (Construct 4) containing three Fc domains and three CD38 binding domains. This construct is formed from a polypeptide containing four Fc domain monomers. The first polypeptide 402 contains three protrusion-containing Fc domain monomers 404, 406, 408 linked by spacers in tandem in tandem. Each of the second, third, and fourth polypeptides (428, 430, 432) is a co-containing Fc domain linked in tandem to a CD38 binding domain (422, 416, 410) containing a V _H domain on the N-terminus. monomers (426, 420, 414). V _L containing domains 424 , 418 , 412 are linked to each V _H domain.
5 is an illustration of an Fc-antigen binding domain construct (Construct 5) containing two Fc domains and three CD38 binding domains. This construct is formed from a polypeptide containing three Fc domain monomers. The first polypeptide (502) contains a CD38 binding domain (510) containing a V _H domain at the N-terminus and two projection-containing Fc domain monomers (508, 506) connected by a spacer in tandem in tandem. . Each of the second and third polypeptides (524, 526) contains co-containing Fc domain monomers (516, 522) linked in tandem to a CD38 binding domain (512, 518) containing a V _H domain on the N-terminus. do. V _L containing domains 504 , 514 , 520 are linked to each V _H domain.
6 is an illustration of an Fc-antigen binding domain construct (Construct 6) containing three Fc domains and four CD38 binding domains. This construct is formed from a polypeptide containing four Fc monomers. The first polypeptide (602) comprises three protrusion-containing Fc domain monomers (606, 608, 610) linked by a spacer in tandem with a CD38 binding domain (612) containing a V _H domain at the N-terminus. contains Each of the second, third, and fourth polypeptides (632, 634, 636) has a co-containing Fc domain linked in tandem to a CD38 binding domain (616, 622, 628) containing a V _H domain on the N-terminus. monomers (618, 624, 630). V _L containing domains 604 , 616 , 622 , 628 are linked to each V _H domain.
7 is an illustration of an Fc-antigen binding domain construct (Construct 7) containing three Fc domains and two CD38 binding domains. This Fc-antigen binding domain construct contains a dimer of two Fc domain monomers (706, 718), wherein both Fc domain monomers are charged amino acids that differ from the WT sequence at their C _H 3 -C _H 3 interface. to promote favorable electrostatic interactions between the two Fc domain monomers. This construct is formed from a polypeptide containing four Fc domain monomers. Each of the two polypeptides (702, 724) contains protrusions-containing protrusions linked by a spacer in tandem to the Fc domain monomers (706, 718) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface. Fc domain monomers (710, 720), and a CD38 binding domain (712, 714) containing a V _H domain on the N-terminus. Each of the third and fourth polypeptides 708, 722 contains a co-containing Fc domain monomer. V _L containing domains 704 and 716 are linked to each V _H domain.
8 is an illustration of an Fc-antigen binding domain construct (Construct 8) containing three Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. Each of the two polypeptides (802, 828) contains protrusions-containing protrusions linked by a spacer in tandem to the Fc domain monomers (810, 816) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface. Fc domain monomers (814, 820). The third and fourth polypeptides (804, 826) each contain co-containing Fc domain monomers (808, 824) linked in tandem to a CD38 binding domain (812, 818) containing a V _H domain at the N-terminus, respectively. do. V _L containing domains 806 , 822 are linked to each V _H domain.
9 is an illustration of an Fc-antigen binding domain construct (Construct 9) containing three Fc domains and four CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. Each of the two polypeptides (902, 936) contains protrusions linked by a spacer in tandem to the Fc domain monomers (910, 924) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface. Fc domain monomers (918, 928), and a CD38 binding domain (908, 920) containing a V _H domain at the N-terminus. The third and fourth polypeptides (904, 934) consist of co-containing Fc domain monomers (916, 932) linked in tandem to a CD38 binding domain (912, 926) containing a V _H domain at the N-terminus. contains V _L containing domains 906 , 914 , 922 , 930 are linked to each V _H domain.
10 is an illustration of an Fc-antigen binding domain construct (Construct 10) containing five Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. Each of the two polypeptides (1002, 1032) contains a different protrusion-containing Fc domain monomer (1014, 1028), an Fc domain monomer (1008, 1022) containing a different charged amino acid from the WT sequence at the C _H 3 -C _H 3 interface. contains protrusion-containing Fc domain monomers (1016, 1030) linked by spacers in tandem in tandem with CD38 binding domains (1006, 1018) containing a V _H domain at the N-terminus. Each of the third, fourth, fifth, and sixth polypeptides (1012, 1010, 1026, 1024) contains a co-containing Fc domain monomer. V _L containing domains 1004 and 1020 are linked to each V _H domain.
11 is an illustration of an Fc-antigen binding domain construct (Construct 11) containing 5 Fc domains and 4 CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (1102, 1148) are linked to other protrusion-containing Fc domain monomers (1120, 1130) and Fc domain monomers (1124, 1126) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface. It contains protrusion-containing Fc domain monomers (1118, 1132) linked by spacers in tandem in a row. Each of the third, fourth, fifth, and sixth polypeptides (1106, 1104, 1144, 1146) is in tandem with a CD38 binding domain (1112, 1122, 1138, 1128) containing a V _H domain at the N-terminus. contains co-containing Fc domain monomers (1116, 1110, 1134, 1140) linked in series with V _L containing domains 1108 , 1114 , 1135 , 1142 are linked to each V _H domain.
12 is an illustration of an Fc-antigen binding domain construct (Construct 12) containing 5 Fc domains and 6 CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (1202, 1256) were linked to another protrusion-containing Fc domain monomer (1226, 1228), an Fc domain monomer (1210, 1244) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface. It contains protrusion-containing Fc domain monomers (1224, 1230) linked by spacers in tandem in tandem, and a CD38 binding domain (1250, 1248) containing a V _H domain at the N-terminus. Each of the third, fourth, fifth, and sixth polypeptides (1206, 1204, 1254, 1252) is in tandem form with a CD38 binding domain (1218, 1212, 1236, 1242) containing a V _H domain at the N-terminus. contains co-containing Fc domain monomers (1222, 1216, 1232, 1238) linked in series with A V _L containing domain ( 1208 , 1214 , 1220 , 1234 , 1240 , 1246 ) is linked to each V _H domain.
13 is an illustration of an Fc-antigen binding domain construct (Construct 13) containing three Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. The two polypeptides (1302, 1324) contain charged amino acids different from the WT sequence at the C _H 3 -C _H 3 interface, linked by spacers in tandem to the protrusion-containing Fc domain monomers (1312, 1316) in tandem. Fc domain monomers (1308, 1318), and a CD38 binding domain (1310, 1314) containing a V _H domain at the N-terminus. The third and fourth polypeptides (1306, 1320) contain a co-containing Fc domain monomer. V _L containing domains 1304 and 1322 are linked to each V _H domain.
14 is an illustration of an Fc-antigen binding domain construct (Construct 14) containing three Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. The two polypeptides (1404, 1426) contain charged amino acids different from the WT sequence at the C _H 3 -C _H 3 interface, linked by spacers in tandem to the protrusion-containing Fc domain monomers (1414, 1418) in tandem. Fc domain monomers (1308, 1318). The third and fourth polypeptides (1402, 1428), respectively, are co-containing Fc domain monomers (1410, 1422) linked in tandem to a CD38 binding domain (1408, 1416) containing a V _H domain at the N-terminus, respectively. contains V _L containing domains 1406 and 1424 are linked to each V _H domain.
15 is an illustration of an Fc-antigen binding domain construct (Construct 15) containing three Fc domains and four CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. The two polypeptides (1502, 1536) contain charged amino acids different from the WT sequence at the C _H 3 -C _H 3 interface, linked by spacers in tandem to the protrusion-containing Fc domain monomers (1518, 1522) in tandem. Fc domain monomers (1512, 1524), and a CD38 binding domain (1514, 1532) containing a V _H domain at the N-terminus. The third and fourth polypeptides (1504, 1534) consist of co-containing Fc domain monomers (1510, 1526) linked in tandem to a CD38 binding domain (1508, 1530) containing a V _H domain at the N-terminus. contains V _L containing domains 1506 , 1516 , 1520 , 1528 are linked to each V _H domain.
16 is an illustration of an Fc-antigen binding domain construct (Construct 16) containing five Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (1602, 1632) are C _H 3-C linked by a spacer in tandem to the protrusion-containing Fc domain monomer (1612, 1622), the second protrusion-containing Fc domain monomer (1614, 1620). It contains an Fc domain monomer (1610, 1624) containing charged amino acids different from the WT sequence at the _H 3 interface, and a CD38 binding domain (1616, 1618) containing a V _H domain at the N-terminus. Each of the third, fourth, fifth, and sixth polypeptides (1608, 1606, 1626, 1628) contains a co-containing Fc domain. V _L containing domains 1604 and 1630 are linked to each V _H domain.
17 is an illustration of an Fc-antigen binding domain construct (Construct 17) containing 5 Fc domains and 4 CD38 binding domains. This construct is formed of a polypeptide containing six Fc monomers. The two polypeptides (1702, 1748) are linked by a spacer in tandem to a protrusion-containing Fc domain monomer (1720, 1730) and a second protrusion-containing Fc domain monomer (1722, 1728) at the N-terminus, Contains Fc domain monomers (1718, 1732) containing charged amino acids different from the WT sequence at the C _H 3 -C _H 3 interface. The third, fourth, fifth, and sixth polypeptides (1706, 1704, 1746, 1744) are in tandem with the CD38 binding domain (1712, 1724, 1738, 1726) containing the V _H domain at the N-terminus. contains co-containing Fc domain monomers (1716, 1710, 1734, 1740) linked in tandem. A V _L containing domain ( 1708 , 1714 , 1736 , 1742 ) is linked to each V _H domain.
18 is an illustration of an Fc-antigen binding domain construct (Construct 18) containing 5 Fc domains and 6 CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (1802, 1856) are C _H 3-C linked by a spacer in tandem to the protrusion-containing Fc domain monomer (1820, 1836), the second protrusion-containing Fc domain monomer (1822, 1834). It contains an Fc domain monomer (1818, 1838) containing charged amino acids different from the WT sequence at the _H 3 interface, and a CD38 binding domain (1826, 1830) containing a V _H domain at the N-terminus. Each of the third, fourth, fifth, and sixth polypeptides (1806, 1804, 1854, 1852) is in tandem with a CD38 binding domain (1812, 1828, 1844, 1850) containing a V _H domain at the N-terminus. contains co-containing Fc domain monomers (1816, 1810, 1840, 1846) linked in series with A V _L containing domain (1808, 1814, 1824, 1832, 1842, 1848) is linked to each V _H domain.
19 is an illustration of an Fc-antigen binding domain construct (Construct 19) containing five Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (1902, 1932) were in tandem with the Fc domain monomers (1908, 1926), which contain different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface, and the projection-containing Fc domain monomers (1916, 1918). It contains protrusion-containing Fc domain monomers (1912, 1930) linked by spacers in tandem in a conformational fashion, and a CD38 binding domain (1914, 1920) containing a V _H domain at the N-terminus. The third and fourth polypeptides (1910, 1928) contain a co-containing Fc domain monomer, and the fifth and sixth polypeptides (1906, 1924) contain a co-containing Fc domain monomer. V _L containing domains 1904 and 1922 are linked to each V _H domain.
20 is an illustration of an Fc-antigen binding domain construct (Construct 20) containing 5 Fc domains and 4 CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (2002, 2048) are an Fc domain monomer (2012, 2030) containing different charged amino acids from the WT sequence at the C _H 3 -C _H 3 interface, and a protrusion-containing Fc domain monomer at the N-terminus ( 2040, 2038) containing protrusion-containing Fc domain monomers (2020, 2022) linked by spacers in tandem in tandem. Each of the third, fourth, fifth, and sixth polypeptides (2006, 2004, 2046, 2044) is in tandem form to a CD38 binding domain (2014, 2042, 2028, 2036) containing a V _H domain at the N-terminus. contains co-containing Fc domain monomers (2018, 2010, 2024, 2032) linked in series with V _L containing domains (2008, 2016, 2026, 2034) are linked to each V _H domain.
21 is an illustration of an Fc-antigen binding domain construct (Construct 21) containing 5 Fc domains and 6 CD38 binding domains. This construct is formed of a polypeptide containing six Fc domain monomers. The two polypeptides (2102, 2156) are Fc domain monomers (2112, 2130) containing different charged amino acids from the WT sequence at the C _H 3-C _H 3 interface, and the other protrusion-containing Fc domain monomers (2144, 2142). It contains protrusion-containing Fc domain monomers (2120, 2122) linked by spacers in tandem in tandem, and CD38 binding domains (2148, 2138) containing a V _H domain at the N-terminus. Each of the third, fourth, fifth, and sixth polypeptides (2106, 2104, 2154, 2152) is in tandem with a CD38 binding domain (2114, 2150, 2128, 2136) containing a V _H domain at the N-terminus. contains co-containing Fc domain monomers (2118, 2110, 2124, 2132) linked in series with V _L containing domains 2108 , 2116 , 2126 , 2134 , 2140 , 2146 are linked to each V _H domain.
22 is three graphs showing the results of CDC, ADCP, and ADCC assays of various anti-CD20 constructs targeting B cells. The first graph shows that the S3Y Fc-antigen binding domain construct can mediate CDC. The middle graph shows that both the SAI and S3Y Fc-antigen binding domain constructs show greater than 100 fold enhanced potency in the ADCP FcγRIIa reporter assay. The third graph shows that the SAI and S3Y Fc-antigen binding domain constructs exhibit enhanced ADCC activity compared to fucosylated mAbs and similar activity to afucosylated mAbs.
23A-23C are schematics of three exemplary methods by which the CD38 binding domain can be linked to the Fc domain of an Fc construct. 23A shows that the heavy chain component of the CD38 binding domain can be expressed as a fusion protein of the Fc chain, and the light chain component can be expressed as a separate polypeptide. 23B shows scFv expressed as a fusion protein of a long Fc chain. 23C shows the heavy and light chain components expressed separately and exogenously added and linked to the Fc-antigen binding domain construct by chemical linkage.
24A shows the amino acid sequence of human IgG1 with EU numbering (SEQ ID NO: 43). The hinge region is double underlined, the CH2 domain is not underlined, and the CH3 domain is underlined.
24B shows the amino acid sequence of human IgG1 with EU numbering (SEQ ID NO: 45). The hinge region lacking E216-C220 (including endpoints) is double underlined, the CH2 domain is not underlined, and the CH3 region is underlined and lacks K447.
Figure 24c shows the amino acid sequence of human IgG1 with EU numbering (SEQ ID NO: 47). The hinge region is double underlined, the CH2 domain is not underlined, and the CH3 domain is underlined and lacks 447K.
24D shows the amino acid sequence of human IgG1 with EU numbering (SEQ ID NO:42). The hinge region lacking E216-C220 (including endpoints) is double underlined, the CH2 domain is not underlined, and the CH3 domain is underlined.
25 shows the results of analysis of dose-dependent binding of anti-CD38 antibodies to those showing relatively high, moderate, and low cell surface CD38 expression among a number of hematologic tumor cell lines. VivoTag645-labeled anti-CD38 antibody binding to living cell surface CD38. Cell surface binding was assessed by FACS analysis.
26A and 26B show the results of an assay showing that the anti-CD38 construct has a similar cell binding profile to an IgG1 anti-CD38 antibody that cross-reacts with human and cyno CD38. (A) Human CD38 expressing Raji tumor cells were transfected with VivoTag645-labeled antibody, SIA-AA-cyno (anti-cyno CD38 mAb), S3Y-AA-cyno-CD38 (construct 13 with cyno CD38 Fab) , anti-CD38 mAb, S3Y-AA-CD38 (construct 13 with anti-CD38 Fab), IgG isotype control and SIF1 control (Fab trimer without Fab region) were incubated for 1 hour at 4°C. . The extent of cell surface binding was assessed by flow cytometry. (b) CHO cells stably expressing cyno CD38 were harvested, and the cell suspension was prepared with VivoTag645-labeled antibody, SIA-AA-cyno (anti-cyno CD38 mAb), S3Y-AA-cyno-CD38 ( Construct 13 with anti-cyno CD38 Fab), anti-CD38 mAb, S3Y-AA-CD38 (Construct 13 with anti-CD38 Fab), IgG isotype control and SIF1 control (Fc trimer without Fab region) sieve) and incubated for 1 hour at 4°C. The extent of cell surface binding was assessed by flow cytometry. Note: The anti-cyno CD38 mAb cross-reactive antibody (S1A-AA-cyno) and S3Y-AA-cyno recognize both human and cyno CD38. In addition, S3Y-AA-cyno CD38 binds to cell surface CD38 better than S1A-AA-cyno (anti-cyno CD38 mAb).
27A to 27D show the evaluation results of CDC activity by anti-CD38 constructs in Daudi cells and Raji cells.
28A and 28B show the evaluation results of tumor cell killing by anti-CD38 constructs in human whole blood. Anti-CD38 construct 13 (S3Y-AA-CD38) shows a highly potent tumor cell killing ability in human whole blood. (A) Effect of anti-CD38 mAb and S3Y-AA-CD38 on killing of Daudi-luciferase tumor cells in human whole blood. (b) Effect of anti-CD38 mAb and S3Y-AA-CD38 on killing of tumor cells in human whole blood. In both (a) and (b), live Daudi-luciferase cells were quantified by adding luciferin substrate and measuring light emission on a luminometer. Calculate % cell death by normalizing the luminescence values of the test samples using a spontaneous lysis control (0% cell lysis) (no antibody addition) and a total lysis control (100% cell lysis). The table shows a comparison of tumor cell death EC50 values from whole blood from three separate human donors. Values represent mean ± SD.
29A to 29C show the evaluation results of endogenous B cell depletion from cynomolgus monkey blood. (a) SIA-AA-cyno (anti-cyno CD38 mAb), S3Y-AA-cyno-011 (construct 13 with cyno CD38 Fab), IgG isotype control and SIF1 on cyno B cells Dose-dependent binding of control (Fc trimer without Fab region). (b) between SIA-AA-cyno, S3Y-AA-cyno-011 (construct 13 with cyno CD38 Fab), IgG isotype control and SIF1 control (Fc trimer without Fab region) A dose-dependent increase in the frequency of no B cell binding. (c) B by SIA-AA-cyno, S3Y-AA-cyno-001 (construct 13 with cyno CD38 Fab), IgG isotype control and SIF1 control (Fc trimer without Fab region) Dose-dependent increase in cell depletion. Treatment with the anti-CD38 construct (S3Y-AA-CD38) resulted in much greater cell depletion than the anti-CD38 mAb. Values are normalized to B cell frequency in untreated controls. (a, b, c) The values plotted in these figures were generated from the same monkey blood donors.
30 shows the results of evaluation of the effect of anti-CD38 constructs in a lymphoma subcutaneous tumor model. SCID mice were inoculated subcutaneously with human lymphoma (Raji) tumor cells. On day 6 after tumor cell transplantation, mice were randomized into treatment groups (n=10 for each) and treated intraperitoneally with 0.5 mL of normal human serum complement (HSC). The next day (on day 7), mice were again injected intraperitoneally with HSC, followed by anti-CD38 mAb (a single IV dose of 5.94 mg/kg), or S3Y-AA-CD38 (a single IV dose of 10 mg/kg), or PBS (single IV injection) intraperitoneally. On day 8, mice received a third HSC IP injection. Tumor growth was routinely monitored by tumor volume measurement. Points marked with ** in the S3Y-AA-CD38 group had a p value of less than 0.0022 compared to the corresponding treatment group.
Figure 31a shows the comparison results of S3Y-AA-CD38 (inverted triangle) and anti-CD38 mAb (circle) for ADCC, ADCP and CDC activity in Daudi cells.
31B shows ADCC, ADCP (measured using a reporter as a surrogate for phagocytosis by macrophages) and S3Y-AA-CD38 for CDC activity against Raji tumor cells resistant to anti-CD38 mAb mediated CDC ( Inverted triangle) and anti-CD38 mAb (circle) comparison results are shown.
Figure 32 shows the results of a study of tumor cell depletion from human whole blood by S3Y-AA-CD38 (inverted triangle) and anti-CD38 mAb (circle).
Figure 33 shows S3Y- in Daudi cells (left panel, relatively high CD38 expression and relatively low CD55 and CD59 expression) and in Raji cells (right panel, relatively low CD38 expression and relatively high CD55 and CD59 expression). Results of studies of complement-mediated cytotoxicity of AA-CD38 (inverted triangle) and anti-CD38 mAb (circle) are shown.
Figure 34a shows the study results of ADCC activity (left panel) and CDC activity (right panel) of S3Y-AA-cyno CD38 (inverted triangle) and anti-cyno CD38 mAb (circle).
Figure 34b shows the study results of ADCC activity (left panel), ADCP activity (middle panel), and CDC activity (right panel) of S3Y-AA-cyno CD38 (inverted triangle) and anti-cyno CD38 mAb (circle). indicates. CDC activity was measured using Raji cells resistant to anti-CD38 mAb mediated CDC.
Figure 35 shows the results of a study of tumor cell depletion by S3Y-AA-cyno CD38 (inverted triangle) and anti-cyno CD38 mAb (circle).
Figure 36 S3Y-AA-cyno CD38 (second bar in each pair) and anti-cyno CD38 mAb in vitro (left panel) and in vivo (right panel). Results of a study comparing B cell depletion by (first bar in each pair) are shown.
Figure 37 shows the results of a study comparing plasma cell depletion by S3Y-AA-CD38 (inverted triangle) and anti-CD38 mAb (circle) in vitro. % plasma cell depletion by either anti-CD38 mAb or S3Y-AA-CD38 in total bone marrow mononuclear cells (BM-MNC) from multiple myeloma patient MM536. Depletion was calculated as the total number of viable CD138 ⁺ cells at each concentration versus baseline values from untreated BM-MNCs.
38A shows the results of a study showing that S3Y-AA-CD38 binding to FcgRIIa, FcgRIIIa and complement is at least 100-fold greater than that of an anti-CD38 mAb.
38B shows that S3Y-AA-CD38 binding to FcγRIIA, FcγRIIIA is enhanced by more than 500-fold over anti-CD38 mAb, and tumor cells S3Y-AA-CD38-opsonized to human complement protein Clq anti-CD38 mAb It shows the results of a study showing that it is 12 times better than that.
39 shows the results of analysis of various constructs for B cell counts in cynomolgus monkeys.
40A and 40B are schematic diagrams of Fc-antigen binding domain constructs containing three Fc domains and two CD38 binding domains. (a) This construct is formed of a polypeptide containing four Fc domain monomers. The two polypeptides contain an Fc domain monomer containing charged amino acids different from the WT sequence at the CH3-CH3 interface, linked by a spacer in tandem in tandem to the protrusion-containing Fc domain monomer, and a VH domain at the N-terminus contains a CD38 binding domain. The third and fourth polypeptides contain a co-containing Fc domain monomer. A VL containing domain is linked to each VH domain. The R292P mutation (indicated by diamonds) was introduced into all six CH2 domains. The six CH2 domains are part of the three Fc domains, formed by the assembly of the four constituent polypeptides. S3Y-CD38 (CC R292P) is an example of this construct. (b) Schematic of the Fc-antigen binding domain construct (Construct 14) containing three Fc domains and two CD38 binding domains. This construct is formed of a polypeptide containing four Fc domain monomers. The two polypeptides contain an Fc domain monomer containing charged amino acids different from the WT sequence at the CH3-CH3 interface, linked by a spacer in tandem to the protrusion-containing Fc domain monomer. Each of the third and fourth polypeptides contain a co-containing Fc domain monomer linked in tandem to a CD38 binding domain containing a VH domain at the N-terminus. A VL containing domain is linked to each VH domain. The R292P mutation (indicated by diamonds) was introduced into all six CH2 domains. The six CH2 domains are part of the three Fc domains, formed by the assembly of the four constituent polypeptides.
Figure 41 shows the results of analysis of human CD38 binding to anti-CD38 antibody and SIF-body as measured by SPR. Binding sensorgrams and 1:1 binding model fit for the anti-CD38 molecule are shown on the left, the Y-axis is % response and the X-axis is time (500 and 1000 s). Kinetic constants and equilibrium constants are shown in the middle and upper right. The stoichiometry of human CD38 binding to an anti-CD38 molecule is shown in the lower right.
Figure 42 shows the results of analysis of human CD38 binding to cyno-CD38 antibody and SIF-body measured by SPR. Binding sensorgrams and 1:1 binding model fit for the cyno-CD38 molecule are shown on the left. Kinetic constants and equilibrium constants are shown in the middle and upper right. The stoichiometry of human CD38 binding to the cyno-CD38 molecule is shown in the lower right.
Figure 43 shows the results of analysis of sinenomolgus CD38 binding to cyno-CD38 antibody, cyno-CD38 SIF-body and CD38 mAb as measured by SPR. Binding sensorgrams and 1:1 binding model fit for the cyno-CD38 molecule are shown in the left panel and bottom middle panel. Binding sensorgrams and 1:1 binding model fit for CD38 mAb are shown in the upper middle panel. Equilibrium constants are shown in the right panel.
44 shows the results of analysis of S3Y-AA-CD38 and S3Y-CC-CD38 for human FcgR versus CD38 mAb using an Fc-gamma receptor homogeneous time-resolved fluorescence (HTRF) assay.
45 shows the results of analysis of CD38 expression on various cell lines.
Figure 46 shows the results of analysis of anti-CD38 mAb and S3Y-CC-CD38 molecules on (a) Daudi cells and (b) Raji cells.
47A and 47B show the results of analysis of S3Y-CC-CD38 for target cell depletion in anti-CD38 mAb-resistant cells. Daudi cells (a) or Raji cells (b) were incubated with human serum complement and S3Y-CC-CD38 or S3Y-CC-CD38 or anti-CD38 mAbs for 2 h at 37 °C in 96-well plates to achieve complement-mediated lysis. promoted Alamar Blue (cell viability reagent) was then added to each well, incubated at 37° C. for 18 hours, and viable cell fluorescence was measured using a fluorometer. % cell lysis = (RFU test - RFU background) × 100 (RFU total cell lysis - RFU background). Values represent mean±SD (n=3).
48A and 48B show the results of analysis of the cytolytic activity of S3Y-CC-CD38A. (A) Primary human NK cells were added to Daudi tumor cells in a ratio of 5:1 in 96-well plates. Then, after the cell mixture was incubated with any one of the drug molecules at 37° C. for 5 hours, dead cells were detected by CytoTox Glo reagent. (b) M2c macrophages were generated by culturing monocytes purified from human PBMC in M-CSF and incubating with IL-10 at 37°C. pHrodo Red-labeled Raji cells were then added to 96-well plates containing macrophage monolayers and then incubated with drug molecules and IL-10 overnight at 37°C in an IncuCyte live cell imaging system. Phagocytosis of pHrodo Red labeled tumor cells by macrophages leads to an increase in captured pHrodo fluorescence by real-time imaging. Values in a and b represent mean±SD (n=3).
Fig. 49 shows the results of analysis of whole blood tumor cell depletion of S3Y-CC-CD38A. Daudi (a) and Raji (b) lymphoma cells labeled with CFSE were added to whole blood and incubated at 37° C. for 18 hours in the presence of anti-CD38A mAb or S3Y-CC-CD38 or S3Y-AA-CD38. RBCs were lysed, and cells were then stained for surface markers, fixed and acquired on a flow cytometer. A decrease in the frequency of CFSE ⁺ (Daudi or Raji) cells in the presence of drug molecules compared to without drug treatment was indicative of selective cell depletion. Values represent mean ± SD.
50 shows the results of analysis of the effects of S3Y-CC-CD38 and S3Y-AA-CD38 on plasma cells from patient biopsies. Bone marrow (BM) biopsies were collected from patients clinically diagnosed with multiple myeloma (MM) according to IMWG criteria. Fresh bone marrow aspirates collected from patients were treated with drug molecules (S3Y and Darzalex) in the presence of autologous plasma from patients to maintain their native microenvironment for 3 hours at 37° C. in a CO ₂ incubator. Samples were then stained and analyzed by flow cytometry for depletion of CD138 ⁺ cells (used as a surrogate marker for CD38 expressing plasma/myeloma cells). Cell depletion was determined using viable CD138 ⁺ cell frequencies out of total single cells, with all relative cell frequencies normalized to baseline parameters of the previous fit, done using cell count as the dependent variable. The highest asymptote of each drug is used as the baseline (set as 100% viable plasma cells).
Figure 51 shows the results of analysis of S3Y-CC-cyno-CD38, S3Y-AA-cyno-CD38, and anti-cyno-CD38 mAb binding to cell surface CD38 on human lymphoma cell lines. Daudi or Raji cells were incubated with VivoTag 645-labeled S3Y or CD38 mAbs at 4° C. for 40 min. Cells were then washed, fixed, and single cell events were acquired on a Cytek Aurora flow cytometer. Data was de-shuffled using SpectroFlo and analyzed in FlowJo. Values represent mean ± SD.
52 shows the results of analysis of the effects of S3Y-CC-cyno-CD38 and anti-cyno-CD38 on Fc effector function. (A) Daudi cells were incubated with human serum complement and S3Y or CD38 mAb for 2 h at 37°C in 96-well plates to promote complement-mediated lysis. Alamar Blue (cell viability reagent) was then added to each well, incubated at 37° C. for 18 hours, and viable cell fluorescence was measured using a fluorometer. % cell lysis = (RFU test - RFU background) × 100 (RFU total cell lysis - RFU background). Values represent mean±SD (n=3). (b) Primary human NK cells were added to Daudi tumor cells in a ratio of 5:1 in 96 well plates. Then, after the cell mixture was incubated with any one of the drug molecules at 37° C. for 5 hours, dead cells were detected by CytoTox Glo reagent. (c) M2c macrophages were generated by culturing monocytes purified from human PBMC in M-CSF and incubating with IL-10 at 37°C. pHrodo Red-labeled Raji cells were then added to 96-well plates containing macrophage monolayers and then incubated with drug molecules and IL-10 overnight at 37°C in an IncuCyte live cell imaging system. Phagocytosis of pHrodo Red labeled tumor cells by macrophages leads to an increase in captured pHrodo fluorescence by real-time imaging. Values in a, b, c represent mean±SD (n=3).
53 shows the results of a single dose PK study. This graph shows the serum concentrations of the indicated drug molecules after a single IV administration in cyno monkeys (n=4/group). Values represent mean ± SEM.
54 shows the results of a single dose study. This graph shows either S3Y-AA-cyno-CD38 (1.7 mg/kg), 3 dose groups of S3Y-CC-cyno-CD38 (0.51, 1.7, 5.1 mg/Kg) or 1 molar equivalent dose of anti-between. B cell changes in blood after one treatment with one dose of either no-CD38-mAb (1.0 mg/Kg), or saline (control) are shown. The percentage change from baseline before dosing in absolute cell counts of CD3 ^- CD20 ⁺ B cells in the peripheral blood of each monkey in the group after intravenous infusion was plotted. Values represent mean ± SEM.
55A and 55B show the results of a multiple repeat dose study. (A) This graph shows B cell changes in blood after treatment with a single dose of S3Y-CC-cyno-CD38 every week. The percentage change from baseline before dosing in absolute cell counts of CD3 ^- CD20 ⁺ B cells in the peripheral blood of each monkey in the group after intravenous infusion was plotted. Values represent mean ± SEM. (b) Tissues were harvested before terminal necropsy after administration of S3Y-CC-cyno-CD38 once a week 4 times. Single cell suspensions were generated from bone marrow and spleen and CD138 ⁺ plasma cell counts were determined by flow cytometry. Values represent mean ± SD.
56A and 56B show analysis of subcutaneous injection of S3Y-CC-cyno-CD38 in cynomolgus monkeys. To evaluate bioavailability, S3Y-CC-cyno-CD38 was administered to cynomolgus monkeys either intravenously as a single dose or by subcutaneous injection. (A) This graph shows the serum concentration of S3Y-CC-cyno-CD38 after single IV or SC administration in cyno monkeys (n=4/group). Values represent mean ± SEM. (b) This graph shows the change of B cells in blood after one treatment with S3Y-CC-cyno-CD38. The percentage change from baseline before dosing in absolute cell counts of CD3 ^- CD20 ⁺ B cells in the peripheral blood of each monkey in the group after intravenous infusion was plotted. Values represent mean ± SEM.

Many therapeutic antibodies recruit elements of the innate immune system through effector functions of the Fc domain, such as antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). function by doing In some cases, the present invention contemplates combining a known single Fc-domain containing therapeutic agent, e.g., the CD38 binding domain of a known therapeutic antibody, with at least two Fc domains to create a novel therapeutic agent with unique biological activity. . In some cases, the novel therapeutic agents disclosed herein have greater biological activity than the biological activity of a known Fc-domain containing therapeutic, eg, a known therapeutic antibody. The presence of at least two Fc domains can enhance effector functions and activate multiple effector functions, such as activating ADCC in combination with ADCP and/or CDC, thereby increasing the efficacy of a therapeutic molecule. It is important to control the number of Fc domains to produce products with consistent biological function. The present invention features a set of Fc engineering tools for assembling discrete sized molecules from a limited number of polypeptide chains by controlling the homodimerization and heterodimerization of peptides encoding the Fc domain. International Patent Application Publication Nos. WO 2015/168643, WO 2017/151971, WO 2017/205436, and WO 2017/205434 disclose Fc engineering tools and methods for assembling molecules having two or more Fc domains, They are incorporated herein by reference in their entirety. The manipulation tools include structural features (eg, glycine linkers) that significantly improve manufacturing results. The properties of these constructs allow for the efficient production of substantially homogeneous pharmaceutical compositions. Such homogeneity in the pharmaceutical composition is desirable to ensure the safety, efficacy, uniformity, and reliability of the pharmaceutical composition. Having a high degree of homogeneity in the pharmaceutical composition not only minimizes potential aggregation or degradation of the pharmaceutical product (eg, degradation products, and/or aggregated products or multimers) caused by unwanted substances, but also , limit off-target and harmful side effects caused by unwanted substances.

As detailed herein, the present inventors have proposed the use of a spacer comprising only glycine residues, the use of a polypeptide sequence in which terminal lysine residues have been removed, and the following two Use of a set of heterodimerization selectivity modules: using an approach comprising (i) a heterodimerization selectivity module with different charge inversion mutations and (ii) a heterodimerization selectivity module with engineered cavities and protrusions; The homogeneity of the composition was improved by engineering the Fc domain components of the Fc-antigen binding domain construct.

We use multiple copies of one long peptide chain containing multiple Fc sequences separated by a linker, and multiple copies of a short chain containing a single Fc sequence, a series of Fc-antigens in which the Fc domains are linked in tandem. Binding domain constructs were designed (Fc-antigen binding domain constructs 1-6; FIGS. 1-6). Heterodimerization mutations were introduced into each Fc sequence to ensure assembly into the desired tandem configuration while minimizing the formation of smaller or larger complexes. Any number of Fc domains can be linked in tandem in this way, which allows the creation of constructs with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains. make it For peptides with N Fc domains, such constructs use 1 to N+1 CD38 binding domains, depending on whether the CD38 binding domain is introduced into a long peptide chain, a short peptide chain, or both, respectively. can be manufactured.

In Fc-antigen binding domain constructs 1 to 6 ( FIGS. 1 to 6 ), the Fc domains were linked with a single branching point between the Fc domains. These constructs contain two copies of a long peptide chain containing multiple Fc sequences separated by a linker, wherein the branching Fc sequence contains the homodimerizing mutation and the unbranched Fc domain is heterologous. contain dimerization mutations. Multiple copies of a short chain comprising a single Fc sequence with mutations complementary to the heterodimerizing mutation in the long chain are used to complete the multimeric Fc scaffold. The heterodimerized Fc domain has a C-terminal end (eg, Fc-antigen binding domain constructs 7-12; FIGS. 7-12), an N-terminal end (eg, Fc-antigen binding domain constructs). 13-18; FIGS. 13-18 ), or both ends of a branching Fc domain (eg, Fc-antigen binding domain constructs 19-21; FIGS. 19-21 ). A plurality of Fc domains in tandem may be linked to either end of a branched Fc domain. CD38 binding domains can be incorporated into long peptide chains, resulting in two CD38 binding domains per assembled protein molecule. Alternatively, the CD38 binding domain is incorporated into a short peptide chain, resulting in N-1 CD38 binding domains per assembled protein molecule, where N is the number of Fc domains in the assembled protein molecule. When a CD38 binding domain is introduced into both short and long peptide chains, the resulting assembled protein molecule contains N+1 CD38 binding domains.

Past engineering efforts for monoclonal antibodies (mAbs) and Fc domains include creating mutations in the Fc domain to enhance binding to FcγRIIIa, thereby enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) responses; Afucosylation of the Fc domain was performed to enhance binding to FcγRIIIa, thereby enhancing the ADCC response.

In contrast to antibodies with mutations in the Fc domain or afucosylation of the Fc domain to enhance binding to FcγRIIIa, the Fc-antigen binding domain constructs disclosed herein are unexpectedly more likely to bind to multiple classes of Fcγ receptors. It is characterized by strong binding and enhanced activity of multiple cytotoxic pathways. The Fc-antigen binding domain constructs of the present invention are capable of enhancing binding to both FcγRIIa and FcγRIIIa compared to their corresponding fucosylated parental monoclonal antibodies and nonfucosylated parental monoclonal antibodies (Example 46) Reference). In addition, the Fc-antigen binding domain constructs of the present invention unexpectedly, in addition to enhancing ADCC pathway responses, complement-dependent cytotoxicity (CDC) pathways and/or antibody-dependent cellular phagocytosis (ADCP) pathways It is characterized by the ability to mediate (see Example 47).

I. Fc domain monomer

The Fc domain monomers include a hinge domain, a C _H 2 antibody constant domain, and a C _H 3 antibody constant domain (eg, a human IgG1 hinge with optional amino acid substitutions, a C _H 2 antibody constant domain, and a C _H 3 antibody constant domain). ) contains at least a portion of The Fc domain monomer may have an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer may also have any immunoglobulin antibody isotype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer may also be hybrid, for example having a hinge and C _H 2 from IgG1 and C _H 3 from IgA, or a hinge and C _H 2 from IgG1 but C from IgG3. has _H 3 . A dimer of Fc domain monomers is an Fc domain (as further defined herein) capable of binding to an Fc receptor, eg FcγRIIIa, a receptor located on the surface of leukocytes. In the present invention, the C _H 3 antibody constant domain of the Fc domain monomer may contain amino acid substitutions at the interface of the C _H 3-C _H 3 antibody constant domain to promote their association with each other. In other embodiments, the Fc domain monomer comprises an additional moiety attached to the N-terminus or C-terminus, such as an albumin-binding peptide or a purified peptide. In the present invention, the Fc domain monomer does not contain antibody variable regions of any type, eg, V _H , V _L , complementarity determining regions (CDRs), or hypervariable regions (HVRs).

In some embodiments, the Fc domain monomers in an Fc-antigen binding domain construct described herein (eg, an Fc-antigen binding domain construct having three Fc domains) are at least 95% identical to the sequence of SEQ ID NO:42. (at least 97%, 99%, or 99.5% identical). In some embodiments, the Fc domain monomers in an Fc-antigen binding domain construct described herein (eg, an Fc-antigen binding domain construct having three Fc domains) are SEQ ID NOs: 44, 46, 48, and 50 to 53, which is at least 95% identical (at least 97%, 99%, or 99.5% identical). In certain embodiments, the Fc domain monomers in the Fc-antigen binding domain construct have a sequence that is at least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 48, 52, and 53. can have

SEQ ID NO: 42

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKKNQVSLTCLDSDNGQPREPQVYTLPPSRDELTKKNQVSLTCLDWSDNGQFQSDIKFSDKVSHEDGSGFFYPHDKFSVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTSKFS

SEQ ID NO: 44

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVVVSLSDKSDELTKNQVVVSLSCAVKGFQPHYPSDIKTSKLPPSRDELTKNQVSLSKAVESNGWFFYPHYPKLKSAVEDKESNGQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGSR

SEQ ID NO: 46

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVVVSLSDSDELTKNQVVSLSDKSDNGQPHYSDIVQVSLPPSRDELTKNQVSLSDKSDNGWPFQSSDITVKSLSCAVKWFFYSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGSRPG

SEQ ID NO: 48

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVVVDVSHEDNGQPHYSDIVQVCTLPPSRDELTKNQVVSLSKAVDNGQPHYPSDITVKVSDKSDNGQPHYSPPKVSDKWSDGFQS

SEQ ID NO: 50

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKKNQVSLAVEVWSDNGQPQSDIKVYTLPPCRDELTKNQVSLAVEVWSDGSPGHSDIKFSKHSVWSDNGGFN

SEQ ID NO: 51

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLKSAVEWSDNGQPQSDIKVYTLPPSRDELTKNQVSLKSAVEWSDPGGFFYPSDIKFSVSLTCLVKGFQN

SEQ ID NO: 52

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLKSAVEWSDNGQPQSDIVYTLPPSRDELTKNQVSLKSAVEWSDPGGFFYPSDIKFSDLKSAVEWSDGGFN

SEQ ID NO: 53

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVVYTLPPCRDKLTKNQSDKFSDKVWSDGGFQSDIKFSDKFSVWSDGSGFFNWYTKSKSVEVVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTSKFS

II. Fc domain

As defined herein, an Fc domain comprises two Fc domain monomers that dimerize by interaction between C _H 3 antibody constant domains. The Fc domain is an Fc receptor, such as an Fc-gamma receptor (i.e. Fcγ receptor (FcγR)), an Fc-alpha receptor (i.e. Fcα receptor (FcαR)), an Fc-epsilon receptor (i.e. Fcε receptor (FcεR)). , and/or form minimal structures that bind to the neonatal Fc receptor (FcRn). In some embodiments, the Fc domains of the invention are Fcγ receptors (eg, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and/or FcγRIV and/or or the neonatal Fc receptor (FcRn).

III. CD38 binding domain

An antigen binding domain comprises one or more peptides or polypeptides that specifically bind to a target molecule. The CD38 binding domain may comprise a CD38 binding domain of an antibody. In some embodiments, the CD38 binding domain may be an antibody or fragment of an antibody-construct, eg, at least a portion of an antibody that binds to a target antigen. The CD38 binding domain may also be a synthetically engineered peptide that specifically binds a target, such as a fibronectin-based binding protein (FN3 monobody).

Fragments Antigen-binding (Fab) fragments are regions on an antibody that bind a target antigen. It consists of one constant domain and one variable domain of each heavy and light chain. The Fab fragment comprises the V _H , V _L , C _H 1 and C _L domains. Each of the variable domains V _H and V _L contains a set of three complementarity determining regions (CDRs) at the amino-terminal end of the monomer. The Fab fragment may have an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. A Fab fragment may also be of any immunoglobulin antibody isotype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4). In some embodiments, a Fab fragment can be covalently attached to a second, identical Fab fragment after protease (eg, pepsin) treatment of an immunoglobulin to form a F(ab′) ₂ fragment. In some embodiments, a Fab may be expressed as a single polypeptide, comprising both variable and constant domains fused, wherein the fusion is, for example, using a linker between these domains.

In some embodiments, only a portion of the Fab fragment may be used as the CD38 binding domain. In some embodiments, only the light chain component of the Fab (V _L + C _L ) may be used, or only the heavy chain component of the Fab (V _H + C _H ) may be used. In some embodiments, a single chain variable fragment (scFv), which is a fusion protein of the V _H and V _L chains of the Fab variable region, can be used. In another embodiment, a linear antibody comprising a pair of tandem Fd segments (V _H -C _H 1 -V _H -C _H 1 ), which together with complementary light chain polypeptides form a pair of CD38 binding regions can be used.

In some embodiments, a CD38 binding domain of the invention comprises, for a target or antigen listed in Table 1, 1, 2, 3, 4, or 1 of the CDR sequences listed in Table 1 for the listed target or antigen; five, or all six, of these CDR sequences as provided in further detail in Table 1 below.

[Table 1]

[Table 2]

Fc-Antigen Binding Domain The CD38 binding domain of Construct 1 (110/104 in FIG. 1 ) may comprise the three heavy chain and three light chain CDR sequences of any one of the antibodies listed in Table 1.

The CD38 binding domain of Fc-antigen binding domain construct 2 (212/204 in FIG. 2) may comprise the three heavy chain and three light chain CDR sequences of any of the antibodies listed in Table 1.

The CD38 binding domains of Fc-antigen binding domain construct 3 (308/316 and 312/318 in FIG. 3) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 4 (410/412, 416/418 and 422/424 in FIG. 4) are the three heavy and three light chain CDRs of any of the antibodies listed in Table 1 sequence may be included.

The CD38 binding domains of Fc-antigen binding domain construct 5 (510/504, 512/514 and 518/520 in FIG. 5) are the three heavy and three light chain CDRs of any of the antibodies listed in Table 1 sequence may be included.

The CD38 binding domains of Fc-antigen binding domain construct 6 (612/604, 614/616, 620/622, and 626/628 in FIG. 6) are the three heavy chains of any of the antibodies listed in Table 1 and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 7 (712/714 and 714/716 in FIG. 7) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 8 (812/806 and 818/822 in FIG. 8) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 9 (908/906, 920/922, 912/914, and 926/930 in FIG. 9) are the three heavy chains of any of the antibodies listed in Table 1 and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 10 (1006/1004 and 1018/1020 in FIG. 10) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 11 (1112/1114, 1122/1108, 1128/1142, and 1138/1136 in FIG. 11) are the three heavy chains of any one of the antibodies listed in Table 1 and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 12 (1218/1220, 1212/1214, 1250/1208, 1248/1246, 1242/1240, and 1236/1234 in FIG. 12) are the antibodies listed in Table 1 3 heavy chain and 3 light chain CDR sequences of any of these.

The CD38 binding domains of Fc-antigen binding domain construct 13 (1310/1304 and 1314/1322 in FIG. 13) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 14 (1408/1406 and 1416/1424 in FIG. 14) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 15 (1508/1506, 1514/1516, 1532/1520, and 1530/1528 in FIG. 15) are the three heavy chains of any of the antibodies listed in Table 1 and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 16 (1616/1604 and 1618/1630 in FIG. 16) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 17 (1712/1714, 1724/1708, 1726/1742, and 1738/1736 in FIG. 17) are the three heavy chains of any of the antibodies listed in Table 1 and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 18 (1812/1814, 1828/1808, 1826/1824, 1830/1832, 1850/1848, and 1844/1842 in FIG. 18) are the antibodies listed in Table 1 3 heavy chain and 3 light chain CDR sequences of any of these.

The CD38 binding domains of Fc-antigen binding domain construct 19 (1914/1904 and 1920/1922 in FIG. 19) will comprise the three heavy and three light chain CDR sequences of any of the antibodies listed in Table 1. can

The CD38 binding domains of Fc-antigen binding domain construct 20 (2014/2016, 2042/2008, 2036/2034, and 2028/2026 in FIG. 20) are the three heavy chains of any one of the antibodies listed in Table 1. and three light chain CDR sequences.

The CD38 binding domains of Fc-antigen binding domain construct 21 (2114/2116, 2150/2108, 2148/2146, 2138/2140, 2136/2134, and 2128/2126 in FIG. 21) are the antibodies listed in Table 1 3 heavy chain and 3 light chain CDR sequences of any of these.

IV. dimerization selectivity module

In the present invention, the dimerization selectivity module comprises selected amino acids or components within the Fc domain monomers that promote the desired pairing of the two Fc domain monomers that will form the Fc domain. Specifically, the dimerization selectivity module is that part of the C _H 3 antibody constant domain of an Fc domain monomer comprising amino acid substitutions located at the interface between the interacting C _H 3 antibody constant domains of the two Fc domain monomers. . In the dimerization selectivity module, amino acid substitutions favor dimerization of the two C _H 3 antibody constant domains as a result of the compatibility of the amino acids selected for such substitution. The eventual formation of advantageous Fc domains is selective compared to other Fc domains formed from Fc domain monomers that either lack a dimerization selectivity module or have incompatible amino acid substitutions within the dimerization selectivity module. Amino acid substitutions of this type can be made using conventional molecular cloning techniques well known in the art, such as QuikChange ^® mutagenesis.

In some embodiments, the dimerization selectivity module comprises an engineered cavity (or “hole” further described herein) within the C _H 3 antibody constant domain. In other embodiments, the dimerization selectivity module comprises engineered protrusions (or “knobs” as further described herein) within the C _H 3 antibody constant domains. To selectively form the Fc domain, two Fc domain monomers with compatible dimerization selectivity modules - eg, a C _H 3 antibody constant domain containing an engineered cavity, and the other an engineered protrusion The containing C _H 3 antibody constant domains combine to form a pair of projections (or “knobs and holes”) into the cavity of the Fc domain monomers. Engineered protrusions and engineered cavities are examples of heterodimerization selectivity modules, in which C _H of Fc domain monomers to promote favorable heterodimerization of two Fc domain monomers with compatible heterodimerization selectivity modules. 3 antibody constant domains. Table 3 lists suitable mutations.

In another embodiment, the heterodimerization involves the use of an Fc domain monomer having a dimerization selectivity module containing a positively charged amino acid substitution and an Fc domain monomer having a dimerization selectivity module containing a negatively charged amino acid substitution. which can be selectively combined to form the Fc domain via favorable electrostatic steering of charged amino acids (described further herein). In some embodiments, the Fc domain monomer may comprise one of the following positively charged and negatively charged amino acid substitutions: K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer containing a positively charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer, containing a negatively charged amino acid substitution, e.g., K370D or K370E, is a charged amino acid through their favorable electrostatic steering, they can be selectively combined to form the Fc domain. In another example, an Fc domain monomer containing E357K and an Fc domain monomer containing K370D can be selectively combined to form an Fc domain via favorable electrostatic steering of charged amino acids. In some embodiments, charge reversal amino acid substitutions may be used as a heterodimerization selectivity module, wherein two Fc domain monomers containing different but compatible charge reversal amino acid substitutions are combined to form a heterodimeric Fc domain. . Table 3 lists various charge reverse dimerization selectivity modules to promote heterodimerization.

In addition to knob and hole mutations and electrostatic steering mutations, there are additional types of mutations that can be used to promote heterodimerization. These mutations are also listed in Table 3.

In another embodiment, the two Fc domain monomers comprise a homodimerization selectivity module containing identical charge inversion mutations at at least two positions within the ring of charged residues at the interface between the C _H 3 domains. The homodimerization selectivity module is a charge inversion amino acid substitution that promotes homodimerization of Fc domain monomers to form a homodimeric Fc domain. By reversing the charge of both members of two or more complementary pairs of residues in the two Fc domain monomers, a mutated Fc domain monomer remains complementary to an Fc domain monomer of the same mutated sequence, but It has lower complementarity to Fc domain monomers that do not have it. In one embodiment, the Fc domain comprises an Fc domain monomer comprising a double mutant K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E. include In another embodiment, the Fc domain comprises an Fc domain monomer comprising a quadruplet mutant combining any pair of double mutants, eg, K409D/D399K/E357K/K370E. Table 4A and Table 4B list the various selectivities that promote homodimerization.

In a further embodiment, the Fc domain monomer containing (i) at least one charge reversal mutation and (ii) at least one engineered cavity or at least one engineered protrusion comprises (i) at least one charge reversal mutation and ( ii) optionally in combination with other Fc domain monomers containing at least one engineered protrusion or at least one engineered cavity to form an Fc domain. For example, by selectively combining an Fc domain monomer containing the charge reversal mutation K370D and engineered co-Y349C, T366S, L368A, and Y407V with other Fc domain monomers containing the charge reversal mutation E357K and engineered protrusions S354C and T366W Fc domains.

The formation of such Fc domains is facilitated by compatible amino acid substitutions in the C _H 3 antibody constant domain. Two dimerization selectivity modules containing incompatible amino acid substitutions, e.g., if both contain engineered protrusions at the C _H 3 -C _H 3 interface, or if both contain identically charged amino acids will not promote the formation of a heterodimeric Fc domain.

Moreover, another method used to promote the formation of an Fc domain with defined Fc domain monomers includes, without limitation, the formation of monomeric α-helices of the leucine zipper for each Fc domain monomer, enabling heterodimer formation. LUZ-Y approach involving C-terminal fusion (US Patent Application Publication No. WO2011034605), as well as heterodimeric Fc domain monomers comprising alternating segments of an IgA C _H 3 sequence and an IgG C _H 3 sequence, respectively. strand-exchange engineered domain (SEED) body approach to generate Fc domains (Davis et al., Protein Eng Des Sel . 23:195-202, 2010).

V. Manipulated Cavities and Manipulated Processes

The use of engineered cavities and engineered protrusions (or "knob-into-hole" strategies) is described by Carter and colleagues (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681, 1998). Knob and hole interactions favor heterodimer formation, whereas knob-knob and hole-hole interactions prevent homodimer formation due to steric collisions and deletions of favorable interactions. The “knob-into-hole” technique is also disclosed in US Pat. No. 5,731,168.

In the present invention, engineered cavities and engineered protrusions are used in the preparation of the Fc-antigen binding domain constructs described herein. Engineered cavities are voids created when an original amino acid in a protein is replaced with a different amino acid with a smaller side chain volume. Engineered protrusions are bumps that are created when an original amino acid in a protein is replaced with a different amino acid with a larger side chain volume. Specifically, the amino acid being replaced is in the C _H 3 antibody constant domain of the Fc domain monomer and is involved in the dimerization of the two Fc domain monomers. In some embodiments, engineered cavities in one C _H 3 antibody constant domain are created to accommodate engineered protrusions in the other C _H 3 antibody constant domain, such that both C _H 3 antibody constant domains are two Fc domain monomers. act as a dimerization selectivity module (eg, a heterodimerization selectivity module) (described above) that promotes or favors the dimerization of In other embodiments, engineered cavities in one C _H 3 antibody constant domain are created to better accommodate the original amino acids in another C _H 3 antibody constant domain. In another embodiment, engineered protrusions in one C _H 3 antibody constant domain are generated to form further interactions with native amino acids in another C _H 3 antibody constant domain.

Engineered cavities can be constructed by replacing amino acids containing larger side chains, such as tyrosine or tryptophan, with amino acids containing smaller side chains, such as alanine, valine, or threonine. Specifically, some dimerization selectivity modules (eg, heterodimerization selectivity modules) (described further above) contain engineered cavities, such as the Y407V mutation, in the C _H 3 antibody constant domain. Similarly, engineered protrusions can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. Specifically, some dimerization selectivity modules (eg, heterodimerization selectivity modules) (described further above) contain engineered protuberances, such as the T366W mutation, in the C _H 3 antibody constant domain. In the present invention, engineered cavities and engineered protrusions are also combined with C _H 3 interdomain disulfide bond manipulation to enhance heterodimer formation. In one example, Fc domain monomers containing engineered co-Y349C, T366S, L368A, and Y407V can be optionally combined with other Fc domain monomers containing engineered protrusions S354C and T366W to form an Fc domain. In another example, an Fc domain monomer containing an engineered cavity with the addition of Y349C and an Fc domain monomer containing an engineered protrusion with the addition of S354C can be optionally combined to form an Fc domain. Other engineered cavities and engineered protrusions in combination with either disulfide bond manipulation or structural calculations (mixed HA-TF) are included in Table 3, without limitation.

Replacing an original amino acid residue in a C _H 3 antibody constant domain with a different amino acid residue may be accomplished by altering the nucleic acid encoding the original amino acid residue. The upper limit on the number of original amino acid residues that can be replaced is the total number of residues at the interface of the C _H 3 antibody constant domain, given that sufficient interactions at the interface are still maintained.

Combination of engineered cavities and engineered protrusions using electrostatic steering

Electrostatic steering can be combined with knob-in-hole techniques to favor heterodimerization between, for example, Fc domain monomers in two different polypeptides. Electrostatic steering, described in more detail below, is the use of favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids within proteins to control the formation of higher order protein molecules. Electrostatic steering can be used to promote either homodimerization or heterodimerization, the latter of which can be usefully combined with knob-in-hole techniques. In the case of heterodimerization, different but compatible mutations are introduced in each Fc domain monomer to be heterodimerized. Accordingly, Fc domain monomers can be modified to include one of the following positively charged and negatively charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D , K409E, K439D, and K439E. For example, one Fc domain monomer, e.g., an Fc domain monomer with cavities (Y349C, T366S, L368A and Y407V), may also contain the K370D mutation and the other Fc domain monomer, e.g. Fc domain monomers with S354C and T366W) may comprise E357K.

More generally, co-mutations (or combinations of mutations), i.e., any of Y407T, Y407A, F405A, Y407T, T394S, T394W:Y407A, T366W:T394S, T366S:L368A:Y407V:Y349C, and S3364H:F405 Can be combined with the electrostatic steering mutation of Table 3, and any of the overhang mutations (or mutation combinations), i.e., T366Y, T366W, T394W, F405W, T366Y:F405A, T366W:Y407A, T366W:S354C, and Y349T:T394F. of can be combined with the electrostatic steering mutations in Table 3.

VI. electrostatic steering

Electrostatic steering is the use of favorable electrostatic interactions between peptides, protein domains, and oppositely charged amino acids within proteins to control the formation of higher order protein molecules. A method of using an electrostatic steering effect to alter the interaction of antibody domains to reduce the formation of homopolymers in favor of the formation of heterodimers in the production of bispecific antibodies is disclosed in US Patent Application Publication No. 2014-0024111. have.

In the present invention, electrostatic steering is used to control dimerization of Fc domain monomers and formation of Fc-antigen binding domain constructs. Specifically, in order to control the dimerization of Fc domain monomers using electrostatic steering, one or more amino acid residues constituting the C _H 3 -C _H 3 interface are converted into positively charged amino acid residues or negatively charged amino acid residues. Alternatively, depending on the particular charged amino acid introduced, the interaction is either electrostatically favorable or unfavorable. In some embodiments, positively charged amino acids such as lysine, arginine, or histidine at the interface are replaced with negatively charged amino acids such as aspartic acid or glutamic acid. In another embodiment, negatively charged amino acids at the interface are replaced with positively charged amino acids. A charged amino acid may be introduced into one or both of the interacting C _H 3 antibody constant domains. By introducing charged amino acids into the interacting C _H 3 antibody constant domains, dimerization selectivity modules (described further above) are generated, which are controlled by electrostatic steering effects resulting from the interactions between the charged amino acids. It is possible to selectively form dimers of Fc domain monomers as described above.

In some embodiments, heterodimerization or homodimerization is performed to generate a dimerization selectivity module comprising an inverted charge, capable of selectively forming dimers of Fc domain monomers as controlled by an electrostatic steering effect. Two Fc domain monomers can be formed selectively via dimerization.

Heterodimerization of Fc domain monomers

Heterodimerization of the Fc domain monomers can be facilitated by introducing different but compatible mutations in the two Fc domain monomers, such as, but not limited to, pairs of charge residues included in Table 3. In some embodiments, the Fc domain monomer may comprise one of the following positively charged and negatively charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer containing a positively charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer, containing a negatively charged amino acid substitution, e.g., K370D or K370E, is a charged amino acid through their favorable electrostatic steering, they can be selectively combined to form the Fc domain. In another example, an Fc domain monomer containing E357K and an Fc domain monomer containing K370D can be selectively combined to form an Fc domain via favorable electrostatic steering of charged amino acids.

For example, in an Fc-antigen binding domain construct having three Fc domains, two of the three Fc domains are produced by heterodimerization of the two Fc domain monomers, as facilitated by electrostatic steering effects. can be formed. "Heterodimeric Fc domain" refers to an Fc domain formed by heterodimerization of two Fc domain monomers, wherein the two Fc domain monomers have different charges that promote the advantageous formation of these two Fc domain monomers. contains a reverse mutation (heterodimerization selectivity module) (see, eg, mutations in Tables 4A and 4B). In an Fc-antigen binding domain construct having three Fc domains - one carboxyl-terminal "stem" Fc domain and two amino-terminal "branched" Fc domains - each amino-terminal "branched" Fc domain is by a heterodimeric Fc domain (also called a "branched heterodimeric Fc domain") (eg, Fc domain monomers 106, 114 or Fc domain monomers 112, 116 in FIG. 1 ). a heterodimeric Fc domain formed; a heterodimeric Fc domain formed by Fc domain monomers 206, 214 or Fc domain monomers 212, 216 in FIG. A branched heterodimeric Fc domain can be formed by an Fc domain monomer containing E357K and another Fc domain monomer containing K370D.

[Table 3]

Note: all residues are numbered according to the EU numbering system (Edelman et al., Proc Natl Acad Sci USA, 63:78-85, 1969)

Homodimerization of Fc domain monomers

Homodimerization of Fc domain monomers can be facilitated by introducing the same electrostatic steering mutation (homodimerization selectivity module) in both Fc domain monomers in a symmetrical manner. In some embodiments, the two Fc domain monomers comprise a homodimerization selectivity module containing identical charge inversion mutations at at least two positions within the ring of charged residues at the interface between the C _H 3 domains. By reversing the charge of both members of two or more complementary pairs of residues in the two Fc domain monomers, a mutated Fc domain monomer remains complementary to an Fc domain monomer of the same mutated sequence, but It has lower complementarity to Fc domain monomers that do not have it. Electrostatic steering mutations that can be introduced into the Fc domain monomer to promote homodimerization are shown in Tables 4A and 4B, without limitation. In one embodiment, the Fc domain comprises two Fc domain monomers, each comprising a double charge inversion mutant (Table 4A and Table 4B), eg, K409D/D399K. In another embodiment, the Fc domain comprises two Fc domain monomers, each comprising a quadruple inversion mutant (Table 4A and Table 4B), eg, K409D/D399K/K370D/E357K.

For example, in an Fc-antigen binding domain construct having three Fc domains, one of the three Fc domains is obtained by homodimerization of the two Fc domain monomers, as facilitated by an electrostatic steering effect. can be formed. A “homodimeric Fc domain” refers to an Fc domain formed by homodimerization of two Fc domain monomers, wherein the two Fc domain monomers have identical charge inversion mutations (e.g., Tables 5 and 6). of mutations). In an Fc-antigen binding domain construct having three Fc domains - one carboxyl-terminal "stem" Fc domain and two amino-terminal "branch" Fc domains - the carboxy-terminal "stem" Fc domain is homologous It may be a dimeric Fc domain (also called a "stem homodimeric Fc domain"). A stem homodimeric Fc domain can be formed by two Fc domain monomers, each containing the double mutant K409D/D399K.

[Table 4A]

[Table 4B]

VII. linker

In the present invention, a linker is used to describe a bond or linkage between polypeptides or protein domains and/or associated non-protein moieties. In some embodiments, the linker is a bond or linkage between at least two Fc domain monomers, and for such binding or linkage, the linker connects the C-terminus of the C _H 3 antibody constant domain of the first Fc domain monomer to the second Fc domain. It is linked to the N-terminus of the hinge domain of the monomer, such that the two Fc domain monomers are linked together in tandem in tandem. In other embodiments, the linker is a bond between an Fc domain monomer and any other protein domain attached thereto. For example, the linker may attach the C-terminus of the C _H 3 antibody constant domain of the Fc domain monomer to the N-terminus of the albumin-binding peptide.

A linker may be a simple covalent bond, such as a peptide bond, a synthetic polymer such as a polyethylene glycol (PEG) polymer, or any kind of bond resulting from a chemical reaction, such as a chemical conjugation. When the linker is a peptide bond, a carboxylic acid group at the C-terminus of one protein domain can react with an amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. Specifically, peptide bonds may be formed from synthetic means through conventional organic chemical reactions well known in the art, or may be produced by natural production from a host cell, where both proteins present in tandem in tandem form For example, a polynucleotide sequence encoding the DNA sequences of two Fc domain monomers is directly transcribed and translated into a contiguous polypeptide encoding both proteins by the necessary molecular machinery, such as DNA polymerase and ribosome, in the host cell. can be

When the linker is a synthetic polymer, such as a PEG polymer, the polymer can be functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the linking ends of the two proteins.

When a linker (other than the peptide bond mentioned above) is prepared from a chemical reaction, a chemical functional group, for example, an amine, carboxylic acid, ester, azide, or other functional group commonly used in the art, is present in one protein. It can be synthetically attached to the C-terminus and to the N-terminus of other proteins, respectively. The two functional groups can then react via synthetic chemical means to form a chemical bond, linking the two proteins together. Such chemical conjugation procedures are routine to those skilled in the art.

spacer

In the present invention, the linker between the two Fc domain monomers is 3 to 200 amino acids (eg, 3 to 200, 3 to 180, 3 to 160, 3 to 140, 3 to 120, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 45, 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200 200, 35 to 200, 40 to 200, 45 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120 to 200, 140-200, 160-200, or 180-200 amino acids). In some embodiments, the linker between two Fc domain monomers is at least 12 amino acids, such as 12-200 amino acids (eg, 12-200, 12-180, 12-160, 12-140, 12 to 120, 12 to 100, 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 12 to 30, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, or 12 or 13 amino acids) (e.g., 14 to 200, 16 to 200, 18 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids). In some embodiments, the linker between the two Fc domain monomers is 12 to 30 amino acids (eg, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids). Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the spacer may contain a motif of GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG (SEQ ID NO: 3), e.g., multiple motifs or repeating motifs. In certain embodiments, the spacer is of GS, e.g., GS, GSGS (SEQ ID NO: 4), GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), or GSGSGSGSGSGS (SEQ ID NO: 8) It may contain from 2 to 12 amino acids comprising a motif. In certain other embodiments, the spacer will contain 3 to 12 amino acids comprising motifs of GGS, for example GGS, GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), and GGSGGSGGSGGS (SEQ ID NO: 11). can In another embodiment, the spacer comprises a motif of GGSG (SEQ ID NO: 2), e.g., GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO: 14), or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 15). 4 to 20 amino acids. In other embodiments, the spacer may contain a motif of GGGGS (SEQ ID NO: 1), for example GGGGSGGGGS (SEQ ID NO: 16) or GGGGSGGGGSGGGGS (SEQ ID NO: 17). In certain embodiments, the spacer is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18).

In some embodiments, the spacer between the two Fc domain monomers contains only glycine residues, e.g., at least 4 glycine residues (eg, 4-200, 4-180, 4-160, 4 to 140, 4 to 40, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6 or 4 or 5 glycine residues) (eg, 4 to 200, 6 to 200, 8 to 200) , 10-200, 12-200, 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200 , 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 glycine residues) contains In certain embodiments, the spacer comprises 4 to 30 glycine residues (eg, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycine residues). In some embodiments, a spacer containing only glycine residues may not be glycosylated (eg, O-linked glycosylation, also referred to as O-glycosylation), or, for example, one or more serine residues (eg, Reduced levels of glycosylation (e.g., reduced levels of O-glycosylation) (e.g., xylose, mannose, sialic acid, reduced levels of O-glycosylation with glycans such as Fuc, and/or galactose (eg, xylose).

In some embodiments, a spacer containing only glycine residues may not be O-glycosylated (eg, O-xylosylated), or, for example, one or more serine residues (eg, SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18) ))), compared to a reduced level of O-glycosylation (eg, a reduced level of O-xylosylation).

In some embodiments, a spacer containing only glycine residues may not undergo proteolysis or reduced compared to a spacer containing, for example, one or more serine residues (eg, SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)). It may have a rate of protein hydrolysis.

In certain embodiments, the spacer will contain a motif of GGGG (SEQ ID NO: 19), e.g., GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21), GGGGGGGGGGGGGGGG (SEQ ID NO: 22), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). can In certain embodiments, the spacer may contain a motif of GGGGG (SEQ ID NO: 24), for example GGGGGGGGGG (SEQ ID NO: 25), or GGGGGGGGGGGGGGG (SEQ ID NO: 26). In certain embodiments, the spacer is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).

In other embodiments, the spacer may also contain amino acids other than glycine and serine, for example GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30), RPACKIPNDLKQKVMNH (SEQ ID NO: 31) , GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 32), AAANSSIDLISVPVDSR (SEQ ID NO: 33), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34).

In certain embodiments of the invention, a 12- or 20-amino acid peptide spacer is used to link the two Fc domain monomers in tandem in tandem, wherein the 12- and 20-amino acid peptide spacers each have the sequence GGGSGGGSGGGS (SEQ ID NO: 35). ) and SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18). In another embodiment, an 18-amino acid peptide spacer consisting of the sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36) may be used.

In some embodiments, the spacer between the two Fc domain monomers is at least 75% identical to the sequence of any one of SEQ ID NOs: 1-36 described above (eg, at least 77%, 79%, 81%, 83%, 85 %, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 99.5% identical). In certain embodiments, the spacer between the two Fc domain monomers is at least 80% identical to the sequence of any one of SEQ ID NOs: 17, 18, 26, and 27 (eg, at least 82%, 85%, 87%, 90 %, 92%, 95%, 97%, 99%, or 99.5% identical). In certain embodiments, the spacer between the two Fc domain monomers is at least 80% identical to the sequence of SEQ ID NO: 18 or 27 (eg, at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 99.5% identical).

In certain embodiments, a linker between the amino-terminus of the hinge of the Fc domain monomer and the carboxy-terminus of the Fc monomer within the same polypeptide (ie, the linker connects the C-terminus of the C _H 3 antibody constant domain of the first Fc domain monomer). linked to the N-terminus of the hinge domain of the second Fc domain monomer, such that the two Fc domain monomers are linked together in tandem in tandem) rather than a covalent bond, at least 3 amino acids (e.g., 3 to 200 amino acids) (For example, 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80, 3-70, 3 to 60, 3 to 50, 3 to 45, 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 or 4, 4 to 200, 5 to 200, 6 to 200, 7 to 200 , 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200 , 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120 to 200, 140 to 200, 160 to 200, or 180 to 200 amino acids)), or at least 12 amino acids, such as 12-200 amino acids (eg, 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, 12 to 14, or 12 to 13 amino acids) (eg, 14 to 200) , 16 to 200, 18 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200 , 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids).

A spacer is also present between the N-terminus of the hinge domain of the Fc domain monomer and the carboxy-terminus of the CD38 binding domain (eg, the CH1 domain of the CD38 heavy chain binding domain or the CL domain of the CD38 light chain binding domain) such that the domains are 3 or more amino acids (e.g., 3 to 200 amino acids (e.g., 3 to 200, 3 to 180, 3 to 160, 3 to 140, 3 to 120, 3 to 100, 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 3 to 45, 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 or 4, 4 to 200, 5 to 200, 6 to 200, 7 to 200, 8 to 200, 9 to 200, 10 to 200, 15 to 200, 20 to 200, 25 to 200, 30 to 200, 35 to 200, 40 to 200, 45 to 200, 50 to 200, 60 to 200, 70 to 200, 80 to 200, 90 to 200, 100 to 200, 120 to 200, 140 to 200, 160 to 200, or 180 to 200 amino acids)), or at least 12 amino acids, such as 12 to 200 amino acids (eg, 12 to 200, 12 to 180, 12 to 160) Dogs, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30 dogs, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, or is 12 to 13 amino acids) (eg, 14 to 200, 16 to 200, 18 to 200, 20 to 200, 30 to 200, 40 to 200, 50 to 200, 60 to 200) amino acids, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids) They are linked by an amino acid spacer containing them.

VIII. Serum protein-binding peptide

Binding to serum protein peptides can improve the pharmacokinetic properties of protein drugs, in particular the Fc-antigen binding domain constructs described herein can be fused with serum protein-binding peptides.

As an example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 37). In some embodiments, the albumin-binding peptide has a sequence that is at least 80% identical (eg, at least 80%, 90%, or 100% identical) to the sequence of SEQ ID NO:37.

In the present invention, the albumin-binding peptide may be attached to the N-terminus or C-terminus of a given polypeptide in the Fc-antigen binding domain construct. In one embodiment, the albumin-binding peptide may be attached to the C-terminus of one or more polypeptides in an Fc construct containing a CD38 binding domain. In another embodiment, the albumin-binding peptide may be fused to the C-terminus of a polypeptide encoding two Fc domain monomers linked in tandem in an Fc construct containing a CD38 binding domain. In another embodiment, the albumin-binding peptide is at the C-terminus of an Fc domain monomer (eg, Fc domain monomers 114, 116 in FIG. 1; Fc domain monomers 214, 216 in FIG. 2). may be attached, the C-terminus being linked to a second Fc domain monomer in a polypeptide encoding two Fc domain monomers linked in tandem in tandem. The albumin-binding peptide may be genetically fused to the Fc-antigen binding domain construct or attached to the Fc-antigen binding domain construct via chemical means, eg, chemical conjugation. If desired, a spacer may be inserted between the Fc-antigen binding domain construct and the albumin-binding peptide. Without wishing to be bound by theory, it is predicted that the inclusion of albumin-binding peptides within the Fc-antigen binding domain constructs of the present invention may lead to prolonged retention of therapeutic proteins through binding to serum albumin.

I. Fc-antigen binding domain constructs

In general, the invention features Fc-antigen binding domain constructs having 2 to 10 Fc domains and one or more CD38 binding domains attached thereto. They may have greater binding affinity and/or avidity for Fc receptors, such as FcγRIIIa, than a single wild-type Fc domain. The present invention addresses the formation of unwanted multimers or aggregates by engineering amino acids at the interface of two interacting C _H 3 antibody constant domains, such that the two Fc domain monomers of the Fc domains selectively form dimers together with each other. How to prevent it is disclosed. The Fc-antigen binding domain construct comprises an even number of Fc domain monomers, wherein each pair of Fc domain monomers forms an Fc domain. The Fc-antigen binding domain construct contains minimally two functional Fc domains and one CD38 binding domain formed from a dimer of four Fc domain monomers. The CD38 binding domain may be linked to the Fc domain using, for example, a linker, a spacer, a peptide bond, a chemical bond or a chemical moiety.

The Fc-antigen binding domain construct can be assembled in many ways. Fc-antigen binding domain constructs can be assembled from asymmetric tandem domains ( FIGS. 1-6 ). An Fc-antigen binding domain construct can be assembled from single branched Fc domains, wherein the branching point is in the N-terminal Fc domain ( FIGS. 7-12 ). An Fc-antigen binding domain construct can be assembled from single branched Fc domains, wherein the branching point is in the C-terminal Fc domain ( FIGS. 13-18 ). An Fc-antigen binding domain construct can be assembled from single branched Fc domains, wherein the branching point is absent in either the N-terminal Fc domain or the C-terminal Fc domain ( FIGS. 19-21 ).

The CD38 binding domain can be linked to the Fc-antigen binding domain construct in a number of ways. The CD38 binding domain can be expressed as a fusion protein of the Fc chain. The heavy chain component of the CD38 binding Fab can be expressed as a fusion protein of the Fc chain, and the light chain component can be expressed as a separate polypeptide ( FIG. 50A ). In some embodiments, an scFv is used as the CD38 binding domain. scFv can be expressed as a fusion protein of a long Fc chain ( FIG. 50B ). In some embodiments, the heavy and light chain components are expressed separately and added exogenously to the Fc-antigen binding domain construct. In some embodiments, the CD38 binding domain is expressed separately and later linked to the Fc-antigen binding domain construct using a chemical bond ( FIG. 50C ).

In some embodiments, one or more Fc polypeptides in the Fc-antigen binding domain construct lack a C-terminal lysine residue. In some embodiments, all Fc polypeptides in the Fc-antigen binding domain construct lack a C-terminal lysine residue. In some embodiments, the absence of a C-terminal lysine in one or more Fc polypeptides in the Fc-antigen binding domain construct results in the absence of a C-terminal lysine in the Fc-antigen binding domain construct (eg, an Fc-antigen binding domain construct with three Fc domains). ), for example, the population of Fc-antigen binding domain constructs having three Fc domains is at least 85%, 90%, 95%, 98%, or 99% homogeneous.

In some embodiments, the first, second, third, fourth, fifth, or sixth polypeptide in the Fc-antigen binding domain construct described herein (eg, polypeptide 102, 112 in FIG. 1 ) , 114), the polypeptides 202, 214, 216, 218 in FIG. 2, the polypeptides 302, 320, 322 in FIG. 3, the polypeptides 402, 428, 430, 432 in FIG. Polypeptides (502, 524, 526) in Figure 6 (602, 632, 634, 636) in Figure 7 (702, 708, 722, 724) in Figure 8 (802, 804, 826, 828), the polypeptides in FIG. 9 (902, 904, 934, 936), the polypeptides in FIG. 10 (1002, 1010, 1012, 1024, 1026, 1032), the polypeptides in FIG. 11 (1102, 1104, 1106) , 1144, 1146, 1148), the polypeptides in FIG. 12 (1202, 1204, 1206, 1252, 1254, 1256), the polypeptides in FIG. 13 (1302, 1306 1320, 1324), the polypeptides 1402, 1404 in FIG. , 1426, 1428), the polypeptides in FIG. 15 (1502, 1504, 1534, 1536), the polypeptides in FIG. 16 (1602, 1606, 1608, 1626, 1628, 1632), the polypeptides in FIG. 17 (1702, 1704, 1706, 1744, 1746, 1748), in Figure 18 (1802, 1804, 1806, 1852, 1854, 1856), in Figure 19 (1902, 1906, 1910, 1924, 1928, 1932), in Figure 20 The N-terminal Asp in one or more of the polypeptides (2002, 2004, 2006, 2044, 2046, 2048) of .

For the exemplary Fc-antigen binding domain constructs described in the Examples herein, Fc-antigen binding domain constructs 1-21 may contain the charge pairs of E357K and K370D in the knob subunit and hole subunit, respectively. .

Any of the exemplary Fc-antigen binding domain constructs described herein (eg, Fc-antigen binding domain constructs 1-21) are antibody-dependent compared to constructs having a single Fc domain and a CD38 binding domain. Constructs that may have enhanced effector function in cytotoxicity (ADCC) assays, antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assays, or have a single Fc domain and a CD38 binding domain It may contain biological activity not exhibited by

X. Host Cells and Protein Production

As used herein, a host cell refers to a vehicle comprising the necessary cellular components, such as organelles, required to express the polypeptides and constructs described herein from the corresponding nucleic acid. The nucleic acid may be included in a nucleic acid vector that can be introduced into a host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells may be of mammalian, bacterial, fungal or insect origin. Mammalian host cells include CHO (or CHO-derived cell lineages, e.g. CHO-K1, CHO-DXB11 CHO-DG44), murine host cells (e.g. NS0, Sp2/0), VERY, HEK (e.g. , HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. A host cell strain that modulates expression of the protein construct, or that modifies and processes protein production in the particular manner desired, can also be selected. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed protein.

For expression and secretion of the protein product from the corresponding DNA plasmid construct, the host cell contains appropriate expression control elements known in the art, including promoters, enhancers, sequences, transcription terminators, polyadenylation sites, and selectable markers. can be transfected or transformed with DNA controlled by Methods for expressing therapeutic proteins are known in the art. See, eg, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology) , Humana Press; 2nd ed. 2004 edition (July 20, 2004)]; Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (June 28, 2012)].

XI. Bifucosylation

Each Fc monomer contains an N-glycosylation site at Asn 297. Glycans can exist in a number of different forms on a given Fc monomer. In compositions containing the antibodies or antigen-binding Fc constructs described herein, glycans can be very heterogeneous and the nature of the glycans present depends, among other things, on generating the antibody or antigen-binding Fc construct. It may depend on the type of cells used, the growth conditions for the cells (including growth media) and post-production purification. In various instances, the constructs or polypeptide complexes described herein or compositions containing the polypeptides are afucosylated, at least to some extent. For example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the glycans (eg, Fc glycans) present in the composition. , 60%, 70%, 80%, 90% or 95% lack fucose residues. Thus, 5% to 60%, 5% to 50%, 5% to 40%, 10% to 50%, 10% to 50%, 10% to 40%, 20% to 50%, or 20% of the glycans to 40% lack fucose residues. An afucosylated composition may be produced, at least to some extent, by culturing the antibody-producing cells in the presence of a 1,3,4-tri-O-acetyl-2-deoxy-2-fluoro-L-fucose inhibitor. can expression in cells with reduced or no expression of FUT8 (e.g., by knocking out FUT8 or reducing expression by RNAi (siRNA, miRNA or shRNA)), and beta-1,4-mannosyl -Relatively afucosylation of the constructs and polypeptides described herein using a variety of other methods, including expression in cells overexpressing the glycoprotein 4-beta-N-acetylglucosaminyltransferase (GnT-III) form can be created.

XII. refine

The Fc-antigen binding domain construct may be prepared by any method known in the art for protein purification, eg, chromatography (eg, ion exchange chromatography, affinity (eg, Protein A affinity) chromatography). chromatography, and size-exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for purification of proteins. For example, the Fc-antigen binding domain construct can be isolated and can be purified (e.g., Process Scale Purification of Antibodies , Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies-Volume I-Production and Purification ). , Kluwer Academic/Plenum Publishers, New York (2004)].

In some cases, the Fc-antigen binding domain construct may be conjugated to one or more purification peptides to facilitate purification and isolation of the Fc-antigen binding domain construct, for example, from a whole cell lysate mixture. . In some embodiments, the purified peptide binds to another moiety with specific affinity for the purified peptide. In some embodiments, those moieties that specifically bind the purified peptide are attached to a solid support, such as a matrix, resin, or agarose bead. Examples of purified peptides that may be linked to the Fc-antigen binding domain construct include, but are not limited to, hexa-histidine peptides, FLAG peptides, myc peptides, and hemagglutinin (HA) peptides. The hexa-histidine peptide (HHHHHH (SEQ ID NO: 38)) binds to a nickel-functionalized agarose affinity column with micromolar affinity. In some embodiments, the FLAG peptide comprises the sequence DYKDDDDK (SEQ ID NO: 39). In some embodiments, the FLAG peptide comprises integer multiples of the sequence DYKDDDDK, eg, 3xDYKDDDDK, in tandem in tandem configuration. In some embodiments, the myc peptide comprises the sequence EQKLISEEDL (SEQ ID NO: 40). In some embodiments, the myc peptide comprises integer multiples of the sequence EQKLISEEDL, eg, 3xEQKLISEEDL, that are in tandem in tandem configuration. In some embodiments, the HA peptide comprises the sequence YPYDVPDYA (SEQ ID NO: 41). In some embodiments, the HA peptide comprises integer multiples of the sequence YPYDVPDYA, eg, 3xYPYDVPDYA, in tandem in tandem configuration. Antibodies that specifically recognize and bind FLAG, myc, or HA purified peptides are well known in the art and are often commercially available. Solid supports (eg, matrix, resin, or agarose beads) functionalized with these antibodies can be used to purify Fc-antigen binding domain constructs comprising FLAG, myc, or HA peptides.

For Fc-antigen binding domain constructs, Protein A column chromatography can be used as the purification process. Protein A ligands interact with the Fc-antigen binding domain constructs via the Fc region, making Protein A chromatography a highly selective capture process capable of removing the majority of host cell proteins. In the present invention, the Fc-antigen binding domain construct can be purified using Protein A column chromatography as described in Example 2.

XIII. Pharmaceutical composition/formulation

The invention features pharmaceutical compositions comprising one or more Fc-antigen binding domain constructs described herein. In one embodiment, the pharmaceutical composition comprises a substantially homogeneous population of Fc-antigen binding domain constructs that are identical or substantially identical in structure. In various examples, the pharmaceutical composition comprises a substantially homogeneous population of any one of Fc-antigen binding domain constructs 1-42.

A therapeutic protein construct of the invention, for example an Fc-antigen binding domain construct described herein (eg, an Fc-antigen binding domain construct having three Fc domains) can be incorporated into a pharmaceutical composition. have. A pharmaceutical composition comprising a therapeutic protein may be formulated by methods known to those skilled in the art. The pharmaceutical composition may be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or other pharmaceutically acceptable liquid. For example, the pharmaceutical composition may contain the Fc-antigen binding domain construct in a pharmaceutically acceptable vehicle or medium such as sterile water for injection (WFI), physiological saline, emulsifying agent, suspending agent, surfactant, stabilizing agent, diluent, binding agent, After suitable blending with excipients, it may be formulated by mixing into unit dosage forms as required in generally accepted pharmaceutical practice. The amount of active ingredient included in a pharmaceutical formulation is such that a suitable dose within the specified range is provided.

Sterile compositions for injection may be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, an isotonic solution or physiological saline containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, optionally with suitable solubilizing agents generally known in the art, for example For example, in combination with alcohols such as ethanol and polyalcohols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as Polysorbate 80™, HCO-50, and the like, as aqueous solutions for injection. Formulation methods for therapeutic protein products are known in the art and are described, for example, in Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2d ed.) Taylor & Francis Group, CRC Press ( 2006)].

XIV. Methods of treatment and administration

The Fc-antigen binding domain constructs described herein can be used to treat a variety of cancers (eg, hematologic malignancies and solid tumors) and autoimmune diseases.

The cancer may be resistant to daratumumab or any other therapeutic anti-CD38 monoclonal antibody therapeutic. The cancer may be selected from gastric cancer, breast cancer, colon cancer, lung cancer, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, NK cell leukemia, NK/T-cell lymphoma, chronic lymphocytic leukemia, plasma cell leukemia, and multiple myeloma. can The constructs can also be used to treat amyloid light chain amyloidosis, Castleman's disease, monoclonal gammopathy of implication (MGUS), biclonal gammopathy of implication, heavy chain disease, solitary plasmacytoma, extramedullary plasmacytoma. In some cases, the constructs can be used to enhance immunomodulatory function against cancer cells by immune complex mediated induction of a prophylactic and/or therapeutic vaccine effect.

The construct may also be used in plasma cell diseases or monoclonal gammopathy, such as light chain deposition disease, membranous proliferative glomerulonephritis (MGRS), autoimmune hemolytic anemia, TEMPI syndrome (Telangiectasia-Erythrocytosis-Monoclonal Gammopathy-Perinephric Fluid Collections-Intrapulmonary Shunting, capillary Angioplasty - Erythrocytosis - Monoclonal Gammaopathy - Perirenal fluid retention - Intrapulmonary shunt), Rheumatoid arthritis, Lupus erythematosus, POEMS Syndrome (Polyneuropathy - Organomegaly - Endocrinopathy - Monoclonal plasmaproliferative disorder - Skin, Polyneuropathy - Organ hypertrophy - Endocrinopathy-monoclonal plasma cell proliferative disorder-skin) and Waldenstrom macroglobulinemia.

The construct may be used in autoantibody-mediated diseases such as myasthenia gravis (MG), MuSK-MG, myocarditis, Lambert-Eaton, myasthenia gravis, neuromuscular dystonia, optic neuromyelitis, narcolepsy, acute motor axonal neuropathy, Guillain-Barré syndrome, Fisher syndrome, acute ataxia neuropathy, paraneoplastic ankylosing syndrome, chronic neuropathy, peripheral neuropathy, acute disseminated encephalomyelitis, multiple sclerosis, Goodpasture syndrome, membranous neuropathy, glomerulonephritis, Alveolar proteinosis, CIPD, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pemphigus vulgaris, pemphigus vulgaris, pemphigus bullosa, pemphigus pregnancy, acquired epidermolysis bullosa, neonatal lupus erythematosus, dermatitis herpeticum, May be used to treat Graves disease, Addson's disease, ovarian insufficiency, autoimmune orchitis, Sjogren's disease, autoimmune gastritis, rheumatoid arthritis, SLE, dry eye disease, vasculitis (acute), carditis, and antibody-mediated rejection have.

The pharmaceutical composition is administered in a manner compatible with the dosage form and in an amount that is therapeutically effective to result in amelioration or recovery of symptoms. The pharmaceutical composition is administered in a variety of dosage forms, for example, intravenous dosage forms, subcutaneous dosage forms, oral dosage forms such as ingestible solutions, drug release capsules, and the like. Appropriate dosages for individual subjects depend on the therapeutic purpose, the route of administration, and the condition of the patient. Generally, the recombinant protein is administered at 1-200 mg/kg, eg 1-100 mg/kg, eg 20-100 mg/kg. Accordingly, it will be necessary for the healthcare provider to adjust the dosage and alter the route of administration as necessary to obtain a titrated and optimal therapeutic effect.

In addition to treating humans, the constructs can be used to treat companion animals, such as dogs and cats, as well as other veterinary subjects.

XV. Complement-dependent cytotoxicity (CDC)

The Fc-antigen binding domain constructs described herein are capable of activating a variety of Fc receptor-mediated effector functions. One component of the immune system is the complement-dependent cytotoxicity (CDC) system, which is part of the innate immune system that enhances the ability of antibodies and phagocytes to clear foreign pathogens. Three biochemical pathways activate the complement system, the classical complement pathway, the alternative complement pathway, and the lectin pathway, all of which involve a set of complex activation and signaling cascades.

In the classical complement pathway, either IgG or IgM triggers complement activation. The C1q protein binds to these antibodies after binding to the antigen, forming the C1 complex. This complex produces C1s esterases, which cleave and activate C4 and C2 proteins into C4a and C4b, and C2a and C2b. The C2a and C4b fragments then form a protein complex called C3 convertase, which cleaves C3 into C3a and C3b, leading to signal amplification and formation of a membrane attack complex.

The Fc-antigen binding domain constructs of the present invention can enhance CDC activity by the immune system.

CDC can be assessed using a colorimetric assay, in which Raji cells (ATCC) are coated with serially diluted antibodies, Fc-antigen binding domain constructs, or IVIg. Human serum complement (Quidel) can be added to all wells at 25% v/v and incubated at 37° C. for 2 hours. After addition of WST-1 cell proliferation reagent (Roche Applied Science), cells can be incubated at 37° C. for 12 hours. The plate can then be placed on a shaker for 2 minutes and the absorbance at 450 nm can be measured.

XVI. Antibody-dependent cell-mediated cytotoxicity (ADCC)

The Fc-antigen binding domain constructs of the present invention can also enhance antibody-dependent cell-mediated cytotoxicity (ADCC) activity by the immune system. ADCC is part of the adaptive immune system in which antibodies bind to surface antigens of foreign pathogens and target them for death. ADCC involves the activation of natural killer (NK) cells by antibodies. NK cells express Fc receptors, which bind to the Fc portion of antibodies such as IgG and IgM. When antibodies bind to the surface of pathogen-infected target cells, they subsequently bind and activate NK cells. NK cells release cytokines such as IFN-γ, and proteins such as perforin and granzyme. Perforin is a pore forming cytolysin that oligomerizes in the presence of calcium. Granzyme is a serine protease that induces programmed cell death in target cells. In addition to NK cells, macrophages, neutrophils and eosinophils can also mediate ADCC.

ADCC can be assessed using a luminescence assay. Thaw human primary NK effector cells (Hemacare) and stand overnight at 37° C. in lymphocyte growth medium-3 (Lonza) at ⁵ ×10 5 cells/mL. The next day, the human lymphoblastic cell line Raji target cells (ATCC CCL-86) were harvested, resuspended in assay medium (RPMI without phenol red, 10% FBSΔ, GlutaMAX™), and in the presence of various concentrations of each probe of interest. plated at 37°C for 30 min. The stationary NK cells are then harvested, resuspended in assay medium, and added to plates containing anti-CD20 coated Raji cells. Plates are incubated for 6 hours at 37° C. with a final ratio of effector-to-target cells of 5:1 (5×10 ⁴ NK cells:1×10 ⁴ Raji).

ADCC activity is determined using the CytoTox-Glo™ Cytotoxicity Assay kit (Promega). The CytoTox-Glo™ assay uses a luminescent peptide substrate to measure dead cell protease activity released by cells that have lost membrane integrity, such as lysed Raji cells. After an incubation period of 6 hours, the prepared reagents (substrates) are added to each well of the plate and placed on a rotary plate shaker for 15 minutes at room temperature. Luminescence is measured using a PHERAstar F5 plate reader (BMG Labtech). Data are analyzed after the readings from the control condition (NK cells + Raji alone) are subtracted from the test condition to remove background.

XVII. Antibody-dependent cellular phagocytosis (ADCP)

The Fc-antigen binding domain constructs of the present invention can also enhance antibody-dependent cellular phagocytosis (ADCP) activity by the immune system. ADCP, also known as antibody opsonization, is the process of marking pathogens for uptake and clearance by phagocytes. Phagocytes are cells that protect the body by ingesting harmful foreign pathogens and dead or dying cells. This process is activated by a pathogen-associated molecular pattern (PAMP), which leads to NF-κB activation. Opsonins such as C3b and antibodies can then be attached to the target pathogen. When the target is coated with opsonins, the Fc domains attract phagocytes through their Fc receptors. Phagocytes then completely enclose the cell, and phagosomes of the ingested material fuse with lysosomes. Subsequent phagolysosomes then proteolytically degrade the cellular material.

ADCP can be assessed using a bioluminescence assay. Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells via FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a). All three receptors can participate in signaling events leading to antibody recognition, immune receptor clustering, and ADCP; Blocking studies suggest that FcγRIIa is the predominant Fcγ receptor involved in this process.

The FcγRIIa-H ADCP Reporter Bioassay is a bioluminescent cell-based assay that can be used to determine the potency and stability of an antibody and other biological properties related to the Fc domain that specifically binds to and activates FcγRIIa. . This assay consists of an engineered Jurkat T cell line expressing a high affinity human FcγRIIa-H variant containing histidine (H) at amino acid 131 and a luciferase reporter driven by an NFAT-response element (NFAT-RE).

When co-cultured with target cells and related antibodies, FcγRIIa-H effector cells bind to the Fc domain of the antibody, resulting in FcγRIIa signaling and NFAT-RE-mediated luciferase activity. The bioluminescent signal is detected and quantified with a luciferase assay and a standard luminophotometer.

Example

The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein may be performed, prepared, and evaluated, and are intended solely to be illustrative of the invention and are intended to help the inventors It is not intended to limit the scope of what is intended to be considered as their disclosure.

Example 1. Design and purification of Fc-antigen binding domain construct 7 with CD38 binding domain

protein expression

To increase folding efficiency, to minimize uncontrolled association of subunits, which can produce unwanted high molecular weight oligomers and multimers, and to be substantially homogeneous (e.g., at least 85%, 90% , 95%, 98%, or 99% homogeneous) of the Fc-antigen binding domain construct to produce a composition for pharmaceutical use. With these objectives in mind, constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Each of Fc-antigen binding domain construct 7 (CD38) comprises two distinct Fc domain monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (SEQ ID NO: ZZ1), and two copies of a short Fc chain (SEQ ID NO: ZZ1); number ZZ2)), and two copies of an anti-CD38 light chain polypeptide (SEQ ID NO: ZZ3). The long Fc chains are in tandem with charge-mutated (K409D/D399K mutation) Fc domain monomers (to promote homodimerization), S354C and T366W protrusions (to promote heterodimerization). -Contains an Fc domain monomer with a formation mutation and an E357K charge mutation, and anti-CD38 VH and CH1 domains at the N-terminus (EU positions 1-220) (Construct 7 (CD38)). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization). The anti-CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain as part of an scFv. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences in Table 7 are encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38), one plasmid encoding a long Fc chain (anti-CD38), and one plasmid encodes a short Fc chain).

[Table 5]

The expressed protein is purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column, followed by further fractionation by ion exchange chromatography. The purified sample is concentrated to approximately 30 mg/mL and sterile filtered through a 0.2 μm filter.

Example 2. Design and purification of Fc-antigen binding domain construct 13 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Each of Fc-antigen binding domain construct 13 (CD38) comprises two distinct Fc domain monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (any of SEQ ID NO: ZZ), and two copies of a short Fc chain (SEQ ID NO: ZZ)), and two copies of an anti-CD38 light chain polypeptide (SEQ ID NO: ZZ). The long Fc chain is in tandem with the Fc domain monomers with S354C and T366W overhang-forming mutations and E357K charge mutations (to promote heterodimerization), the charge (to promote homodimerization) -Contains a mutated (K409D/D399K mutation) Fc domain monomer, and anti-CD38 VH and CH1 domains at the N-terminus (EU positions 1-220) (Construct 13 (CD38)). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization). Anti-CD38 light chain and anti-CD38 VH and CH1 are taken from anti-CD38 monoclonal antibody. Constructs with this light chain and anti-CD38 VH and CH1 are designated by the abbreviation CD38. Related constructs can be generated using anti-CD38 light chains and anti-CD38 VH and CH1 taken from fully human monoclonal antibodies that cross-react with CD38 expressed by cynomolgus monkeys. These constructs are denoted by the abbreviation Cyno. The CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain as part of an scFv. Different versions of construct 13 can be prepared using an anti-CD38 heavy chain, each version comprising a different sized glycine spacer (G4, G10, G15 or G20 linker) between the Fc domain monomers in the long Fc chain polypeptide. have The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for each of the following constructs are encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38), one plasmid encoding a long Fc chain (anti-CD38), and one plasmid encoding a short Fc chain):

[Table 6]

The expressed protein was purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column, then the purified sample was concentrated to approximately 30 mg/mL, and 0.2 μm filter. sterilized through filtration.

Example 3. Design and purification of Fc-antigen binding domain construct 1

Unbranched constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 1 ( FIG. 1 ) comprises two distinct Fc domain monomer containing polypeptides (a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. the long Fc chain is two Fc domain monomers in tandem in tandem configuration, wherein each Fc domain monomer has at least one protrusion-forming mutation selected from Table 3 (to promote heterodimerization) (e.g. engineered protrusions prepared by introducing the S354C and T366W mutations), and optionally having one or more charge reversal mutations (e.g., E357K) selected from Table 4A or Table 4B - and at the N-terminus It has a CD38 binding domain. The CD38 binding domain may be expressed as part of the same amino acid sequence as the long Fc chain (eg, to form an scFv). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation (e.g., Y349C, T366S, L368A, and Y407V mutations) selected from Table 3 (to promote heterodimerization); and optionally, an Fc domain monomer having a charge inversion mutation (eg, K370D) selected from Table 4A or Table 4B. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. In this example, and in each of the following examples for Fc-antigen binding domain constructs 2-42, the cell may contain a third plasmid expressing an antibody variable light chain.

The expressed protein is purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column. The captured Fc-antigen binding domain construct is washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3 . The eluate is rapidly neutralized by the addition of 1 M TRIS (pH 7.4) and sterile filtered through a 0.2 μm filter. Proteins are further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated using 50 mM MES, pH 6 (buffer A), and samples were run in a step gradient using 50 mM MES, 400 mM sodium chloride, pH 6 as elution buffer (buffer B). elute After ion exchange, the target fraction is buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Concentrate the sample to approximately 30 mg/mL and filter sterile through a 0.2 μm filter.

Samples are denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95° C. for 10 minutes. Samples are run on Criterion TGX dye-free gels (4-15% polyacrylamide, Bio-Rad). The protein bands are visualized with UV light or Coomassie blue staining. The gel is imaged with a ChemiDoc MP imaging system (Bio-Rad). Quantification of bands is performed using Imagelab 4.0.1 software (Bio-Rad).

Example 4. Design and purification of Fc-antigen binding domain construct 2

Unbranched constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 2 ( FIG. 2 ) comprises two separate Fc monomer containing polypeptides (a long Fc chain and 3 copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains three Fc domain monomers present in tandem in tandem with the CD38 binding domain at the N-terminus, wherein each Fc domain monomer has at least one protrusion-forming mutation selected from Table 3 ( engineered protrusions prepared by introducing, for example, S354C and T366W mutations), and optionally one or more charge inversion mutations selected from Table 4A or Table 4B (eg E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 5. Design and purification of Fc-antigen binding domain construct 3

Constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 3 ( FIG. 3 ) comprises two separate Fc monomer containing polypeptides (a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains two Fc domain monomers that are in tandem in tandem configuration, wherein each Fc domain monomer carries at least one protrusion-forming mutation selected from Table 3 (e.g., the S354C and T366W mutations). engineered protrusions prepared by introducing them, and optionally, one or more charge inversion mutations selected from Table 4A or Table 4B (eg E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 6. Design and purification of Fc-antigen binding domain construct 4

Constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 4 ( FIG. 4 ) comprises two separate Fc monomer containing polypeptides (a long Fc chain and 3 copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains three Fc domain monomers that are in tandem in tandem configuration, wherein each Fc domain monomer carries at least one protrusion-forming mutation selected from Table 3 (e.g., the S354C and T366W mutations). engineered protrusions prepared by introducing them, and optionally, one or more charge inversion mutations selected from Table 4A or Table 4B (eg E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer with a selected charge inversion mutation (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 7. Design and purification of Fc-antigen binding domain construct 5

Constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 5 ( FIG. 5 ) comprises two distinct Fc monomer containing polypeptides (a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains two Fc domain monomers that are in tandem in tandem with a CD38 binding domain at the N-terminus, wherein each Fc domain monomer has at least one protrusion-forming mutation selected from Table 3 ( engineered protrusions prepared by introducing, for example, S354C and T366W mutations), and optionally one or more charge inversion mutations selected from Table 4A or Table 4B (eg E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer with a selected charge inversion mutation (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 8. Design and purification of Fc-antigen binding domain construct 6

Constructs formed from asymmetric tandem Fc domains are prepared as described below. Fc-antigen binding domain construct 6 ( FIG. 6 ) comprises two distinct Fc monomer containing polypeptides (a long Fc chain and 3 copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains three Fc domain monomers present in tandem in tandem with the CD38 binding domain at the N-terminus, wherein each Fc domain monomer has at least one protrusion-forming mutation selected from Table 3 ( engineered protrusions prepared by introducing, for example, S354C and T366W mutations), and optionally one or more charge inversion mutations selected from Table 4A or Table 4B (eg E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer with a selected charge inversion mutation (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 9. Design and purification of Fc-antigen binding domain construct 7

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 7 ( FIG. 7 ) comprises two separate Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B , and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 10. Design and purification of Fc-antigen binding domain construct 8

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 8 ( FIG. 8 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B contains The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 11. Design and purification of Fc-antigen binding domain construct 9

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 9 ( FIG. 9 ) comprises two separate Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B , and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 12. Design and purification of Fc-antigen binding domain construct 10

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 10 ( FIG. 10 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain consists of two Fc domain monomers in tandem in tandem configuration - each Fc domain monomer comprising an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B; Engineered protrusions prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, S354C and T366W mutations) present in tandem in tandem, and optionally selected from Table 4A or Table 4B having one or more charge reversal mutations (eg, E357K) that is, and contains a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 13. Design and purification of Fc-antigen binding domain construct 11

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 11 ( FIG. 11 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain contains two Fc domain monomers that are in tandem in tandem configuration, wherein each Fc domain monomer has a charge inversion mutation selected from Table 4A or Table 4B at the N-terminus (e.g., K409D/ An engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (e.g., S354C and T366W mutation) in tandem with the Fc domain monomer having the D399K mutation, and optional , having one or more charge inversion mutations (eg, E357K) selected from Table 4A or Table 4B. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and an antigen-binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 14. Design and purification of Fc-antigen binding domain construct 12

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 12 ( FIG. 12 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain consists of two Fc domain monomers in tandem in tandem configuration - each Fc domain monomer comprising an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B; Engineered protrusions prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, S354C and T366W mutations) present in tandem in tandem, and optionally selected from Table 4A or Table 4B having one or more charge reversal mutations (eg, E357K) that is, and contains a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and an antigen-binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 15. Design and purification of Fc-antigen binding domain construct 13

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 13 ( FIG. 13 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. The long Fc chain comprises an engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, the S354C and T366W mutation), and optionally one or more protrusions selected from Table 4A or Table 4B. An Fc domain monomer having a charge reversal mutation (eg, K409D/D399K mutation) selected from Table 4A or 4B, which is in tandem with the Fc domain monomer having a charge inversion mutation (eg, E357K) , and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 16. Design and purification of Fc-antigen binding domain construct 14

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 14 ( FIG. 14 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. The long Fc chain is an engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, S354C and T366W mutations), and optionally from Table 4A or Table 4B at the N-terminus. A charge reversal mutation selected from Table 4A or Table 4B (eg, K409D/D399K mutation) in tandem with an Fc domain monomer having one or more charge reversal mutations selected (eg, E357K) It contains an Fc domain monomer with The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 17. Design and purification of Fc-antigen binding domain construct 15

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 15 ( FIG. 15 ) comprises two separate Fc monomer containing polypeptides (two copies of a long Fc chain and two copies of a short Fc chain) and a light chain polypeptide. The long Fc chain comprises an engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, S354C and T366W mutations), and optionally one or more protrusions selected from Table 4A or Table 4B. An Fc domain monomer having a charge reversal mutation (eg, K409D/D399K mutation) selected from Table 4A or Table 4B, which is in tandem with the Fc domain monomer having a charge inversion mutation (eg, E357K) , and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 18. Design and purification of Fc-antigen binding domain construct 16

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 16 ( FIG. 16 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain comprises an engineered protrusion prepared by introducing at least one protrusion-forming mutation (eg, S354C and T366W mutation) selected from Table 3, respectively, and optionally one selected from Table 4A or Table 4B Having a charge reversal mutation selected from Table 4A or Table 4B (eg K409D/D399K mutation) in tandem with two Fc domain monomers having one or more charge reversal mutations (eg E357K) It contains an Fc domain monomer, and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 19. Design and purification of Fc-antigen binding domain construct 17

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 17 ( FIG. 17 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain comprises an engineered protrusion prepared by introducing at least one protrusion-forming mutation (eg, S354C and T366W mutation) selected from Table 3, respectively, and optionally, Table 4A or Table 4B at the N-terminus. A charge reversal mutation selected from Table 4A or Table 4B (eg K409D/D399K) present in tandem with two Fc domain monomers having one or more charge reversal mutations (eg E357K) selected from mutations) containing Fc domain monomers. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 20. Design and purification of Fc-antigen binding domain construct 18

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 18 ( FIG. 18 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. The long Fc chain comprises an engineered protrusion prepared by introducing at least one protrusion-forming mutation (eg, S354C and T366W mutation) selected from Table 3, respectively, and optionally one selected from Table 4A or Table 4B Having a charge reversal mutation selected from Table 4A or Table 4B (eg K409D/D399K mutation) in tandem with two Fc domain monomers having one or more charge reversal mutations (eg E357K) It contains an Fc domain monomer, and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 21. Design and purification of Fc-antigen binding domain construct 19

Constructs formed from a single branched Fc domain lacking either the N-terminal Fc domain or the C-terminal Fc domain with a branching point are prepared as described below. Fc-antigen binding domain construct 19 ( FIG. 19 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B , and an engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (eg, S354C and T366W mutations), and optionally one or more charge inversion mutations selected from Table 4A or Table 4B (eg, E357K) containing other Fc domain monomers, and a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. contains an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D). The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 22. Design and purification of Fc-antigen binding domain construct 20

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 20 ( FIG. 20 ) comprises two distinct Fc monomer containing polypeptides (2 copies of a long Fc chain and 4 copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B , and an engineered protrusion prepared by introducing at least one protrusion-forming mutation (e.g., S354C and T366W mutation) selected from Table 3, and optionally, Table 4A or Table 4B at the N-terminus. contains other Fc domain monomers with one or more charge inversion mutations (eg, E357K). The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 23. Design and purification of Fc-antigen binding domain construct 21

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Fc-antigen binding domain construct 21 ( FIG. 21 ) comprises two distinct Fc monomer containing polypeptides (two copies of a long Fc chain and four copies of a short Fc chain) and a light chain polypeptide. at least one protrusion selected from Table 3, wherein the long Fc chain is in tandem with an Fc domain monomer having a charge reversal mutation (eg K409D/D399K mutation) selected from Table 4A or Table 4B- An Fc domain monomer having an engineered protrusion prepared by introducing a formation mutation (e.g., S354C and T366W mutations), and optionally, one or more charge inversion mutations (e.g. E357K) selected from Table 4A or Table 4B , an engineered protrusion prepared by introducing at least one protrusion-forming mutation selected from Table 3 (e.g., S354C and T366W mutations), and optionally one or more charge reversal mutations selected from Table 4A or Table 4B ( eg, E357K), and contains a CD38 binding domain at the N-terminus. The short Fc chain is an engineered cavity prepared by introducing at least one co-forming mutation selected from Table 3 (e.g., Y349C, T366S, L368A, and Y407V mutations), and optionally from Table 4A or Table 4B. an Fc domain monomer having one or more selected charge inversion mutations (eg, K370D), and a CD38 binding domain at the N-terminus. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc chains are encoded by two separate plasmids. The expressed protein is purified as in Example 3.

Example 24. CDC, ADCP and ADCC activation by Fc-antigen binding domain constructs

Three assays are used to test activation of the CDC, ADCP and ADCC pathways by parental mAbs and various Fc-antigen binding domain constructs. Four constructs containing CDRs from Gazyva (obinutuzumab), an anti-CD20 mAb, are generated. Both fucosylated anti-CD20 mAb and non-fucosylated anti-CD20 mAb, as well as S3Y-AA-CD20 (structure of Construct 13, as described in FIG. 13 , Example 2) and SAI-AA-CD20 ( Structure of Construct 7, as described in FIG. 7 , Example 1) Fc-antigen binding domain constructs were also prepared.

The CDC assay is performed as follows:

1. The target cells used in the anti-CD20 CDC assay are Raji cells (ATCC CCL-86). Raji cells (CD20 expressing tumor cells) were resuspended at 6×10 ⁵ cells/ml in X-VIVO 15 medium. Cells were then transferred to 96 well flat bottom assay plates in a volume of 100 μl per well (6×10 ⁴ cells/well).

2. Anti-CD20 mAb and Fc-antigen binding domain constructs were diluted to 3.33 μM in X-VIVO 15 medium. Serial 1:3 dilutions were then performed for each molecule in 1.5 ml polypropylene tubes to obtain 11 point serial dilutions.

3. Transfer each dilution of these molecules at 50 μl/well to the appropriate wells in the assay plate. Immediately after transfer to the assay plate, 50 μl of normal human serum complement was added to each well.

4. Assay plates were incubated at 37° C. and 5% CO ₂ for 2 hours. After 2 hours of incubation, 20 μl of WST-1 proliferation reagent was added to each well of the assay plate. The plate was returned to the 37° C., 5% CO ₂ incubator and left for 14 hours.

5. After 14 h of incubation, the plate was shaken on a plate shaker for 1 min and the absorbance of the wells was immediately determined at 450 nm with a 600 nm calibration using a spectrophotometer.

In the CDC assay in which the target cell was Raji ( FIG. 22 , left panel), S3Y-AA-CD20 (construct 13 with anti-CD20 Fab) was able to mediate cytotoxicity, whereas the other constructs were not.

ADCP assays were performed as follows:

The FcγRIIa-H ADCP Reporter Bioassay, Complete Kit (Promega Cat # G9901) can be used to determine the potency and stability of the antibody and other biological properties relating to the Fc domain that specifically binds to and activates FcγRIIa. It is a bioluminescent cell-based assay that can This assay consisted of an engineered Jurkat T cell line expressing a high-affinity human FcγRIIa-H variant containing histidine (H) at amino acid 131 and a luciferase reporter driven by an NFAT-response element (NFAT-RE). When co-cultured with target cells and related antibodies, FcγRIIa-H effector cells, upon binding to the Fc domain of the antibody, result in FcγRIIa signaling and NFAT-RE-mediated luciferase activity. Bioluminescence signals were detected and quantified using a Bio-Glo™ Luciferase Assay System and a luminophotometer. Increasing concentrations of anti-CD20 mAb and construct 7 (with anti-CD20 Fab) or construct 13 (with anti-CD20 Fab) were combined with (2:1 ratio) Raji target cells and FcγRIIa-H effector cells incubated. After 6 hours of incubation at 37° C., Bio-Glo™ reagent was added and luminescence was measured on a PHERAstar FS instrument. Data were fitted to a 4PL curve using GraphPad Prism software ( FIG. 22 , middle panel). Both the S3I-AA-CD20 construct (construct 7 with anti-CD20 Fab) and S3Y-AA-CD20 construct (construct 13 with anti-CD20 Fab) were more than 100 fold greater than the anti-CD20 mAb. It showed improved potency (EC50).

ADCC assays were performed as follows:

Human primary NK effector cells were thawed and left overnight at 37° C. in lymphocyte growth medium-3 (Lonza) at 5×10 ⁵ cells/mL. The next day, Raji cells were harvested, resuspended in assay medium (RPMI without phenol red, 10% FBS, GlutaMAX™) and plated at 37° C. for 30 minutes in the presence of various concentrations of each molecule of interest. The stationary NK cells were then harvested, resuspended in assay medium and added to plates containing anti-CD20 coated Raji cells. Plates were incubated for 6 hours at 37° C. with a final ratio of effector-to-target cells of 5:1 (5×10 ⁴ NK cells:1×10 ⁴ Raji cells).

ADCC activity was determined using the CytoTox-Glo™ Cytotoxicity Assay Kit (Promega). The CytoTox-Glo™ assay uses a luminescent peptide substrate to measure dead cell protease activity released by cells that have lost membrane integrity, such as lysed Raji cells. After an incubation period of 6 hours, the prepared reagents (substrates) were added to each well of the plate and placed on a rotary plate shaker for 15 minutes at room temperature. Luminescence was measured using a PHERAstar F5 plate reader (BMG Labtech). Data were analyzed after the readings from the control condition (NK cells + Raji alone) were subtracted from the test condition to remove background. (FIG. 47, right panel). Both the S3I construct (construct 7 with anti-CD20 Fab) and the S3Y construct (construct 13 with anti-CD20 Fab) showed enhanced cytotoxicity compared to fucosylated mAbs, and nonfucosylated mAbs showed similar cytotoxicity compared to

Example 25. Experimental assays used to characterize Fc-antigen binding domain constructs

Peptide and Glycopeptide Liquid Chromatography-MS/MS

Proteins were diluted to 1 μg/μL in 6 M guanidine (Sigma). Dithiothreitol (DTT) was added at a concentration of 10 mM under denaturing conditions at 65° C. for 30 minutes to reduce disulfide bonds. After cooling on ice, samples were incubated with 30 mM iodoacetamide (IAM) in the dark for 1 h to alkylate (carbamidomethylate) the free thiols. The protein was then dialyzed across a 10 kDa membrane into 25 mM ammonium bicarbonate buffer, pH 7.8 to release IAM, DTT and guanidine. removed. Proteins were digested with trypsin in a Barocycler (NEP 2320; Pressure Biosciences, Inc.). The pressure was cycled between 20,000 psi and ambient pressure at 37° C. for a total of 30 cycles per hour. LC-MS/MS analysis of the peptides was performed on an Ultimate 3000 (Dionex) chromatography system and a Q-Exactive (Thermo Fisher Scientific) mass spectrometer. Peptides were separated on a BEH PepMap (Waters) column using 0.1% FA in water and 0.1% FA in acetonitrile as mobile phases. A single xylosylated linker peptide was targeted based on a double charged ion (z=2) m/z 842.5 using a quadrupole isolation width of ± 1.5 Da.

Intact mass spectrometry

Proteins were diluted to a concentration of 2 μg/μL in running buffer consisting of 78.98% water, 20% acetonitrile, 1% formic acid (FA), and 0.02% trifluoroacetic acid. Size exclusion chromatographic separations were performed on two Zenix-C SEC-300s (Sepax Technologies, Newark, Del.) (2.1×350 mm) in tandem for a total length column length of 700 mm. Proteins were eluted from the SEC column using the running buffer described above at a flow rate of 80 μL/min. Mass spectra were acquired on a QSTAR Elite (Applied Biosystems) Q-ToF mass spectrometer operated in positive ion mode. The neutral mass under the individual size fractions was deconvoluted using Bayesian peak deconvolution by summing the spectra across the full width of the chromatographic peak.

Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) Assay

Samples were diluted to 1 mg/mL and mixed with HT Protein Express Denaturing Buffer (PerkinElmer). The mixture was incubated at 40° C. for 20 minutes. Samples were diluted with 70 μL of water and transferred to 96 well plates. Samples were analyzed by a Caliper GXII instrument (PerkinElmer) equipped with an HT Protein Express LabChip (PerkinElmer). Fluorescence intensity was used to calculate the relative abundance of variants of each size.

Non-reducing SDS-PAGE

Samples were denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95° C. for 10 minutes. Samples were run on Criterion TGX dye-free gels (4-15% polyacrylamide, Bio-Rad). Protein bands were visualized by UV illumination or Coomassie blue staining. The gel was imaged with a ChemiDoc MP imaging system (Bio-Rad). Quantification of bands was performed using Imagelab 4.0.1 software (Bio-Rad).

complement dependent cytotoxicity (CDC)

CDC was assessed as described above in Example 24.

Example 26. Design and purification of Fc-antigen binding domain construct 4 with CD38 binding domain

protein expression

Constructs formed from asymmetric tandem Fc domains were prepared as described below. Each of Fc-antigen binding domain construct 4 (CD38) comprises two distinct Fc domain monomer containing polypeptides (a long Fc chain (SEQ ID NO: 66), and three copies of an anti-CD38 Fc chain (SEQ ID NO: 68)) and 3 copies of the anti-CD38 light chain polypeptide (SEQ ID NO: 49). The long Fc chain contains three Fc domain monomers that are in tandem in tandem configuration, wherein each Fc domain monomer has an E357K charge mutation (to promote heterodimerization) and S354C and T366W protrusion-forming mutations . The short Fc chain consists of an Fc domain monomer with a K370D charge mutation (to promote heterodimerization) and Y349C, T366S, L368A, and Y407V co-forming mutations, and anti-CD38 VH and CH1 domains at the N-terminus ( EU positions 1-220) (Construct 4 (CD38)). The CD38 light chain can also be expressed fused to the N-terminus of a short Fc chain as part of an scFv. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 7 are encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38), one plasmid encoding a long Fc chain, and one plasmid encoding a short Fc chain (anti-CD38):

[Table 7]

The expressed protein is purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column. The captured Fc-antigen binding domain construct is washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3 . The eluate was rapidly neutralized by the addition of 1 M TRIS (pH 7.4) and sterile filtered through a 0.2 μm filter. Proteins are further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES pH 6 (buffer A) and the samples eluted with a step gradient using 50 mM MES, 400 mM sodium chloride pH 6 as elution buffer (buffer B). After ion exchange, target fractions were buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Concentrate the sample to approximately 30 mg/mL and filter sterile through a 0.2 μm filter.

Example 27. Design and purification of Fc-antigen binding domain construct 8 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain were prepared as described below. Each of Fc-antigen binding domain construct 8 (CD38) comprises two distinct Fc domain monomer containing polypeptides (two copies of a long Fc chain (SEQ ID NO: 69), and two copies of an anti-CD38 short Fc chain (SEQ ID NO: 69). 68)), and a copy of the anti-CD38 light chain polypeptide (SEQ ID NO: 49). The long Fc chains are in tandem with the Fc domain monomers with charge inversion mutations K409D and D399K (to promote homodimerization), S354C and T366W protrusion-forming (to promote heterodimerization). It contains an Fc domain monomer with a mutation and an E357K charge mutation. The short Fc chain consists of an Fc domain monomer with a K370D charge mutation (to promote heterodimerization) and Y349C, T366S, L368A, and Y407V co-forming mutations, and anti-CD38 VH and CH1 domains at the N-terminus ( EU positions 1-220) (Construct 8 (CD38)). The CD38 light chain can also be expressed fused to the N-terminus of a short Fc chain as part of an scFv. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 8 are encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38), one plasmid encoding a long Fc chain, and one plasmid encoding a short Fc chain (anti-CD38):

[Table 8]

The expressed protein is purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column. The captured Fc-antigen binding domain construct is washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3 . The eluate is rapidly neutralized by the addition of 1 M TRIS (pH 7.4) and sterile filtered through a 0.2 μm filter. Proteins are further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column is pre-equilibrated with 50 mM MES pH 6 (buffer A) and the sample is eluted with a step gradient using 50 mM MES, 400 mM sodium chloride pH 6 as elution buffer (buffer B). After ion exchange, the target fraction is buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Concentrate the sample to approximately 30 mg/mL and filter sterile through a 0.2 μm filter.

Example 28. Design and purification of Fc-antigen binding domain construct 9 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain were prepared as described below. Fc-antigen binding domain construct 9 (CD38) consists of two distinct Fc domain monomer containing polypeptides: two copies of an anti-CD38 long Fc chain (SEQ ID NO: 54), and two copies of an anti-CD38 short Fc chain. (SEQ ID NO: 68)), and a copy of the anti-CD38 light chain polypeptide (SEQ ID NO: 49). The long Fc chains are in tandem with the Fc domain monomers with charge inversion mutations K409D and D399K (to promote homodimerization), S354C and T366W protrusion-forming (to promote heterodimerization). Fc domain monomer with mutation and E357K charge mutation, and anti-CD38 VH and CH1 domains at the N-terminus (EU positions 1-220) (Construct 9 (CD38)). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization), and an anti-CD38 heavy chain at the N-terminus ( Construct 9 (CD38)). The CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain and/or a short Fc chain as part of an scFv. The DNA sequence was optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 9 were encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38) and one plasmid encoding a long Fc chain (anti-CD38) encoding, one plasmid encoding a short Fc chain (anti-CD38):

[Table 9]

Example 29. Design and purification of Fc-antigen binding domain construct 10 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with a branching point in the N-terminal Fc domain are prepared as described below. Each of Fc-antigen binding domain construct 10 (CD38) comprises two distinct Fc domain monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (SEQ ID NO: 71), and four copies of a short Fc chain (SEQ ID NO: 71). 63)), and a copy of the anti-CD38 light chain polypeptide (SEQ ID NO: 49) for each. The long Fc chain is two Fc domain monomers in tandem in tandem, wherein each Fc domain monomer is in tandem with the Fc domain monomers having charge inversion mutations K409D and D399K (to promote homodimerization) with S354C and T366W overhang-forming mutations and E357K charge mutations (to promote heterodimerization), and anti-CD38 VH and CH1 domains at the N-terminus (EU positions 1-220) Contains (Construct 10 (CD38)). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization). The anti-CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain as part of an scFv. The DNA sequence was optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 10 were encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38) and one plasmid encoding a long Fc chain (anti-CD38) , and one plasmid encodes a short Fc chain):

[Table 10]

The expressed protein was purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column. The captured Fc-antigen binding domain construct was washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3 . The eluate is rapidly neutralized by the addition of 1 M TRIS (pH 7.4) and sterile filtered through a 0.2 μm filter. Proteins are further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column is pre-equilibrated with 50 mM MES pH 6 (buffer A) and the sample is eluted with a step gradient using 50 mM MES, 400 mM sodium chloride pH 6 as elution buffer (buffer B). After ion exchange, the target fraction is buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Concentrate the sample to approximately 30 mg/mL and filter sterile through a 0.2 μm filter.

Example 30. Design and purification of Fc-antigen binding domain construct 16 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with a branching point in the C-terminal Fc domain are prepared as described below. Each of Fc-antigen binding domain construct 16 (CD38) comprises two distinct Fc domain monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (SEQ ID NO: 73), and four copies of a short Fc chain (SEQ ID NO: 73); 63)), and three copies of an anti-CD38 light chain polypeptide (SEQ ID NO: 49) for each. The long Fc chain is in tandem with two Fc domain monomers in tandem, each with S354C and T366W protrusion-forming mutations and E357K charge mutations (to promote heterodimerization), ( Fc domain monomer with charge inversion mutations K409D and D399K to promote homodimerization, and anti-CD38 VH and CH1 domains at the N-terminus (EU positions 1-220) (construct 10 (CD38) )). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization). The anti-CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain as part of an scFv. The DNA sequence is optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. The DNA plasmid construct is transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 11 are encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38) and one plasmid encoding a long Fc chain (anti-CD38) , and one plasmid encodes a short Fc chain):

[Table 11]

The expressed protein is purified from the cell culture supernatant by protein A-based affinity column chromatography using a Poros MabCapture A (Life Technologies) column. The captured Fc-antigen binding domain construct is washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3 . The eluate is rapidly neutralized by the addition of 1 M TRIS (pH 7.4) and sterile filtered through a 0.2 μm filter. Proteins are further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column is pre-equilibrated with 50 mM MES pH 6 (buffer A) and the sample is eluted with a step gradient using 50 mM MES, 400 mM sodium chloride pH 6 as elution buffer (buffer B). After ion exchange, the target fraction is buffer exchanged into PBS buffer using a 10 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Concentrate the sample to approximately 30 mg/mL and filter sterile through a 0.2 μm filter.

Example 31. Design and purification of Fc-antigen binding domain construct 19 with CD38 binding domain

protein expression

Constructs formed from a single branched Fc domain with no branching points in either the N-terminal Fc domain or the C-terminal Fc domain were prepared as described below. Fc-antigen binding domain construct 19 (CD38) consists of two distinct Fc domain monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (SEQ ID NO: 75), and four copies of a short Fc chain (SEQ ID NO: 75) 63)), and a copy of the anti-CD38 light chain polypeptide (SEQ ID NO: 49) for each. The long Fc chain is in tandem with the Fc domain monomers with S354C and T366W overhang-forming mutations and E357K charge mutations (to promote heterodimerization), the charge (to promote homodimerization) Fc domain monomers with S354C and T366W overhang-forming mutations and E357K charge mutations (to promote heterodimerization) in tandem with the Fc domain monomers with reverse mutations K409D and D399K, and the N-terminus contains the anti-CD38 VH and CH1 domains (EU positions 1-220) in (Construct 19 (CD38)). The short Fc chain contains Fc domain monomers with Y349C, T366S, L368A, and Y407V co-forming mutations and K370D charge mutations (to promote heterodimerization). The anti-CD38 light chain can also be expressed fused to the N-terminus of a long Fc chain as part of an scFv. The DNA sequence was optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. The following amino acid sequences for each construct in Table 12 were encoded by three separate plasmids (one plasmid encoding a light chain (anti-CD38) and one plasmid encoding a long Fc chain (anti-CD38) , and one plasmid encodes a short Fc chain):

[Table 12]

Example 32. Binding of anti-CD38 constructs to human tumor cell lines and stable cell lines expressing human and cynomolgus monkey CD38

Tumor cell suspensions in medium containing 10% FBS were incubated with increasing concentrations of VivoTag645-labeled anti-CD38 antibody at 4° C. for 1 hour. Cells were then washed in cold buffer and suspended in FACS buffer. The labeled cell suspension was then read on the APC channel on a BD FACS Verse flow cytometer. Unlabeled cells were used to gate live cell populations. FlowJo software was used to calculate geometric mean fluorescence intensity (gMFI) values from the gated populations. The results of this analysis are presented in FIG. 25 .

Raji cells were used to evaluate the dose-dependent relative binding of the parental IgGl anti-CD38 antibody and the corresponding anti-CD38 construct. Since the anti-CD38 mAb (which is the source of Fab for various anti-CD38 Fc constructs) does not cross-react with monkey CD38, we present a surrogate anti-CD38 human monoclonal that reacts with cynomolgus monkey CD38. A surrogate anti-CD38 construct 13 (S3Y-AA-cyno CD38) was generated using the same Fab sequence, which reacts with an IgG1 antibody (S1A-AA-cyno CD38) and cynomolgus monkey CD38; It was used to assess CDC activity in the presence of cynomolgus monkey serum complement and to determine the pharmacodynamic response of targeting endogenous cynomolgus monkey CD38 in non-human primate whole blood. The results of these binding studies are presented in FIG. 26 .

Example 33. CDC activity of anti-CD38 constructs

The ability of anti-CD38 antibodies and anti-CD38 Fc constructs to promote cell death of CD38-expressing tumor cell lines (Daudi and Raji) was assessed by an in vitro CDC assay. Human serum complement was used as the complement source. RPMI-1640 medium containing 0.1% BSA was used as a buffer to prepare cell suspensions, antibodies, and serum dilutions. CD38 positive tumor cells were first washed in buffer and resuspended at a density of 10 ⁶ cells/ml. In a typical assay, 50 μl of antibody or anti-CD38 Fc construct, 50 μl of diluted complement (5X dilution), and 50 μl of cell suspension (50,000 cells/well) are added to a flat-bottom tissue culture 96-well plate. did. The mixture was then incubated for 2 h at 37° C. in a 5% CO ₂ incubator to promote complement-mediated cell lysis. Then, 50 μL of Alamar Blue was added to each well and incubated at 37° C. for 18 hours. Fluorescence was read using a 96 well fluorometer with excitation at 530 nm and emission at 590 nm.

This assay was performed using Daudi cells and Raji cells in the presence of human or cyno serum complement to evaluate the relative CDC-mediated tumor cell lysis induced by either the anti-CD38 mAb or the anti-CD38 construct. Results presented in Table 13 are expressed in relative fluorescence units (RFU) proportional to the number of viable cells. The activity of various mutants was investigated by plotting the % CDC activity against the log of the Ab concentration (final concentration before adding Alamar Blue). % CDC activity was calculated as follows: % CDC activity = (RFU test - RFU background) x 100 (RFU in total cell lysis - RFU background). Values represent mean±SD from representative experiments (from separate experiments n=3). This study showed that the anti-CD38 construct had greater potency (maximum tumor cell killing) and potency than the anti-CD38 mAb in anti-CD38 mAb-CDC sensitive cells (Daudi) as well as in anti-CD38 mAb-CDC resistant cells (Raji). prove that it represents The term 'anti-CD38 mAb-sensitive' or 'anti-CD38 mAb-resistance' refers to sensitivity or resistance to anti-CD38 mAb mediated target cell lysis in a cell-based CDC assay.

[Table 13]

Cynomolgus monkey CD38 cross-reactive anti-CD38 construct 13 (S3Y-AA-cyno CD38) was associated with the corresponding mAb S1A-AA-cyno (anti-CD38) in inducing CDC in both susceptible and resistant tumor cells. -Cyno CD38 mAb) showed significantly higher potency and efficacy. This assay was performed in a similar manner to that described above, except that Daudi tumor cells and monkey serum complement (Figure 27A), Raji tumor cells and monkey serum complement (Figure 27B), Daudi tumor cells and human serum complement (Figure 27C), Raji tumors Cellular and human serum complement ( FIG. 27D ) were used. CDC of these constructs The activity is presented in Table 14, which shows the efficacy of S3Y-AA-cyno CD38 versus S1A-AA-cyno (anti-cyno CD38 mAb) in inducing CDC on Daudi and Raji cells and It shows a significant improvement in potency.

[Table 14]

Example 34. Antibody-Dependent Cellular Phagocytosis (ADCP) Activation by Anti-CD38 Fc Constructs

Monocytes were isolated from human whole blood and allowed to differentiate into macrophages by treating them with human M-CSF and IL-10 in 6 well plates. These adherent macrophages were then detached using chilled PBS+2 mM EDTA for subsequent seeding into assay wells. 2×10 ⁵ macrophages were seeded in RPMI-1640 medium containing 2% ultra-low FBS in 96 well flat bottom plates. Plates were briefly centrifuged and incubated at 37° C. for 1 hour to attach macrophages to the bottom of 96-well plates. Raji tumor cells were stained with Calcein-AM followed by a 3:1 effector (macrophage):target (tumor cell) ratio in the presence of serial dilutions of anti-CD38 mAb or various anti-CD38 constructs. Phagocytes were added onto the plate. The plates were then incubated for 2 hours at 37° C. in a CO ₂ incubator. Supernatants were collected in V-bottom 96 well plates. Adherent cells were collected by detachment using chilled PBS containing 2 mM EDTA. Cells from the supernatant and detached adherent cells were pooled together. These cells were then stained with these antibodies by incubating with anti-CD11b APC and anti-CD19 BV421 antibodies at 4° C. for 1 hour. Labeled cell suspensions were read on a FACS Verse flow cytometer. Double positive macrophages negative for surface CD19 staining (CD11b ⁺ /calcein-AM ⁺ ) were considered phagocytic events. The results are presented in Table 15, which shows the superior potency of anti-CD38 constructs in inducing phagocytosis of Raji cells opsonized by primary human macrophages.

[Table 15]

Example 35. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activation by Anti-CD38 Fc Constructs

Raji cells were suspended in RPMI medium containing 10% trough IgG FBS in 96 well plates at a concentration of 5000 cells/50 μL medium/well. Samples were then incubated with increasing concentrations of antibody and construct (10 uL/well) at 25° C. for 15 minutes. Primary human NK cells (effector cells) were added at an effector to target ratio of 5:1. The effector and target cell mixes were then incubated for 5 hours at 37° C. in a 5% CO ₂ incubator. CytoTox Glo reagent (50 μL) was added and the plate was incubated at 25° C. for 15 min to label dead cells. The samples were then read on a Pherastar luminometer to determine the luminescent signal from dead cells. Our results demonstrate a 5- to 7-fold higher potency of the S3Y (construct 13) molecule compared to the anti-CD38 mAb in inducing ADCC. As shown in Table 15 below, anti-CD38-construct 13 demonstrated superior potency than anti-CD38 mAb in inducing primary human NK cell-mediated ADCC against Raji tumor cells. After the target cells and effector cells were treated with drug molecules at 37° C. for 5 hours, dead cells were detected by CytoTox Glo reagent. assay controls, spontaneous release control (target cells alone); antibody-free control; NK cells alone + antibody; IgGk isotype control.

[Table 15]

Example 36: Tumor Cell Killing by Anti-CD38 Construct in Human Whole Blood

Daudi cells were suspended in 50 μl of medium (RPMI-1640 + 10% ultra-low IgG FBS) and seeded into each well of a 96 well plate. 50 μL of human whole blood or ACK-lysed human whole blood cells (no serum and no RBCs) were added to the tumor cell suspension. Then 50 μL of antibody and anti-CD38 construct dilutions (in RPMI-1640 medium + 10% FBS) were added. Samples were mixed and then incubated in a CO ₂ incubator at 37° C. for 4 hours. After incubation, the remaining live Daudi cells were assessed by adding 50 μL of freshly prepared luciferin solution (stock concentration, 50 mg/mL). The plate was then placed on a plate shaker for 5 minutes. Luminescence emitted from live Daudi-luciferase cells was read using a Pherastar luminometer.

The results presented in Figure 28 show that anti-CD38 construct 13 (S3Y-AA-CD38) is 10- to 36-fold more potent than anti-CD38 mAb in killing target cells in human whole blood collected from three separate donors. suggest that However, for RBC lysis and washed whole blood, no tumor cell depletion was observed for either the anti-CD38 mAb or the anti-CD38 construct. Tumor cell depletion was restored when RBC lysed and washed human whole blood cells were supplemented with autologous serum prepared from the same donor, which was used to promote anti-CD38 mAb and anti-CD38 construct-induced tumor cell death in whole blood. suggest the role of serum proteins in

Example 37: Depletion of Endogenous CD38 Expressing B Cells from Monkey Whole Blood

Cyno whole blood was separately transfected with each VivoTag645-labeled molecule (SIF1, IgG isotype control, S1A-AA-cyno-001 (anti-cyno CD38 mAb), anti-cyno CD38 mAb) with a cocktail of cell surface marker antibodies. Serial dilutions of Construct 13 S3Y-AA-Cyno-001) were mixed. Blood samples were then incubated at 4° C. for 30 minutes to determine cell surface binding, or individually incubated for 3 hours at 37° C. in a CO ₂ incubator to determine the effect of treatment on cell depletion. After these treatments, the RBCs were dissolved by mixing the samples with cold ammonium chloride solution. The samples were then washed and resuspended in buffer containing 1% paraformaldehyde and FACS analysis was performed the next day. CD38 ⁺ B cell populations were evaluated based on CD38-binding and binding-frequency data. The frequency of CD38 ⁺ B cell types was measured to determine depletion due to treatment with construct molecules for 3 hours. B cell depletion was observed in a dose-dependent manner at doses above 10 nM (1 Log nM) for anti-CD38 construct 13 (S3Y-AA-cyno-001). Depletion begins to appear between 100 and 1000 nM (2-3 Log nM). A greater depletion was observed for anti-cyno CD38 construct 13 (S3Y-AA-cyno-001) compared to the anti-cyno CD38 mAb (S1A-AA-cyno-001).

Example 38: In Vivo Lymphoma Model

The effects of agents on disease progression and therapeutic response were evaluated in a subcutaneous tumor model for human lymphoma by tumor volume measurement. CB17-severe combined immunodeficiency (SCID) mice (female, 6-7 weeks old, average body weight 20 g, strain 236 from Charles River Laboratories) were housed at the Momenta animal shelter for 48 hours prior to use according to the IACUC protocol. Water and food were provided ad libitum. All experiments were approved by the Institutional Animal Ethics Committee. Mice were checked daily for signs of discomfort and general appearance. For the subcutaneous tumor xenograft model, 5 x 10 ⁶ human Burkitt's lymphoma Raji cells suspended in high concentration Matrigel were injected subcutaneously into the right flank of mice. Tumor volumes were measured twice a week until tumors reached approximately 250 mm 3 (approximately from days 6 to 7), at which point mice were assigned to treatment groups (8 mice/group). Mice in all three groups received 0.5 mL of normal human serum complement the day before treatment, immediately prior to intravenous treatment injection (with PBS, anti-CD38 mAb, or S3Y-AA-CD38), and one day after treatment. Intraperitoneal injection. Body weight and tumor volume were recorded twice a week. Tumors were measured daily when the volume approached 2000 mm 3 . All animals were observed daily; Affected animals were euthanized according to the IACUC protocol. The results shown in Figure 30 suggest that anti-CD38 construct 13 (S3Y-AA-CD38) is more efficacious than the anti-CD38 mAb in this human lymphoma mouse model when treatment is given in the presence of human serum complement. do.

Example 39: Fc Domain in Construct Retains Binding Similar to Binding of Fc Domain in Antibody to Fc Gamma Receptor

Anti-CD20 constructs and anti-CD38 constructs were used to determine whether various combinations of homodimerization mutations, heterodimerization mutations, polypeptide linkers, and Fab domains affected binding to Fc gamma receptors. evaluated. 1:1 binding to CD64 (Fc gamma receptor I) was evaluated using surface plasmon resonance (SPR). Constructs were captured on the chip surface and binding to soluble receptors was measured to ensure 1:1 binding. In this format, binding valency is the most sensitive readout for alterations in Fc function; Kinetic constants and equilibrium constants are insensitive to alterations in a subset of Fc domains.

cell culture

The DNA sequence was optimized for expression in mammalian cells and cloned into pcDNA3.4 mammalian expression vector. DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. Antibodies were expressed from two different plasmids: one encoding the heavy chain and the second encoding the light chain. SIF-bodies were expressed from three separate plasmids: in most cases one plasmid encoding the antibody light chain and one plasmid long Fc containing the CH1-VH FAB portion attached to the amino-terminal Fc. chain, and the third plasmid encoded a short Fc chain. The exceptions were the S3A and S3W SIF-bodies. For S3W, one plasmid encoded an antibody light chain, a second plasmid encoded a long chain containing two Fc domains, and a third plasmid encoded a single Fc chain containing a CH1-VH FAB portion. For S3A, one plasmid encoded an antibody light chain, a second plasmid encoded a long Fc chain containing a CH1-VH FAB portion attached to an amino-terminal Fc, and one plasmid encoded a CH1-VH FAB portion It also encodes a short Fc chain containing

protein purification

The expressed protein was purified from the cell culture supernatant by Protein A-based affinity column chromatography using a Poros MabCapture A column. The captured SIF-body construct was washed with phosphate buffered saline (PBS, pH 7.0) after loading and further washed with an intermediate wash buffer 50 mM citrate buffer, pH 5.5 to remove further process-related impurities. . The bound SIF-body material was eluted with 100 mM glycine, pH 3, and the eluate was rapidly neutralized by addition of 1 M TRIS, pH 7.4, then centrifuged and sterile filtered through a 0.2 μm filter.

Proteins were further fractionated by ion exchange chromatography using Poros XS resin. The column was pre-equilibrated with 50 mM MES pH 6 (buffer A) and samples were diluted (1:3) in equilibration buffer for loading. Samples were eluted using a linear gradient of 12-15 CV from 50 mM MES (100% A) to 400 mM sodium chloride (pH 6) (100% B) as elution buffer. All fractions collected during elution were analyzed by analytical size exclusion chromatography (SEC), and target fractions were pooled to generate purified SIF-body material.

After ion-exchange, the pooled material was buffer exchanged into 1X-PBS buffer using a 30 kDa cutoff polyether sulfone (PES) membrane cartridge on a tangential flow filtration system. Samples were concentrated to approximately 10-15 mg/mL and sterile filtered through a 0.2 μm filter.

physicochemical analysis

Analytical size exclusion chromatography (SEC) was used to evaluate the purity of protein A followed by the pooled ion-exchange fractions, and the final purified material.

The purified material was diluted to 1 mg/ml with 1X-PBS and using a Zenix SEC-300 (4.6 x 300 mm, 3 μm, 300 Å, Sepax, Cat. #213300-4630) as analytical column. Analysis was performed on an Agilent 1200 system with UV and FLD detectors.

The column was equilibrated using 100 mM sodium phosphate, 200 mM arginine, 300 mM sodium chloride (pH = 6.7) with 0.05% w/v sodium azide buffer at 0.3 ml/min for 1 hour prior to analysis. Injection amount: approximately 10 to 15 ul; column temperature: 300°C; FLD with UV detection at 280 nm and excitation at 280 mm and emission at 330 nm; Total deployment time: 15 minutes.

The size purity results are shown in Table 16. All materials showed only low levels of high order species (HOS).

[Table 16]

binding analysis

Binding experiments were performed on a Biacore T200 instrument (GE Healthcare) using a CM3 series S sensor chip. For valency analysis of FcgR binding, native protein A was directly immobilized through amine coupling. Ligand was diluted in running buffer and captured. Six-point serial dilutions of human recombinant CD32a or CD64 (R&D Systems) were flowed over the captured ligand. The valency of each ligand was calculated as follows:

Ligand valency = Rmax / [(MW analyte / MW ligand) * level of ligand capture].

Results from analysis of CD64 binding to anti-CD20 constructs are shown in Table 17. In all cases, CD64 avidity was equal to the number of Fc domains, indicating that all Fc domains were functional for binding CD64. Control compounds identical in sequence to S3Y-AA-OBI and S3Y-AA-AVE but lacking the Fab domain bound CD64 comparable to those constructs, indicating that inclusion of the Fab domain resulted in binding to Fc receptors. prove that it has not been changed.

[Table 17]

Example 40: Constructs Bind More Vigorously to Cell Surface Fc Gamma Receptors

The relative binding of the constructs to cell surface CD32a was assessed in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay (CisBio) using an anti-CD20 construct. Assay reagents were prepared according to the manufacturer's instructions. A Freedom EVOware 150 automated liquid handling device (Tecan) was used to generate 10-point, 3-fold serial dilutions for each sample, which were added to cells bearing labeled receptors. The labeled competitor antibody was then added and the plate incubated at room temperature. The assay plates were read at 665 and 620 nm using a PHERAstar fluorescence reader (BMG Labtech GmbH). Log-transformed sample concentrations were plotted against the corresponding HTRF signal ratio (665 nm/620 nm). A 4-parameter nonlinear regression analysis (least squares fit) was performed on the XY-plots to calculate the EC50 of unlabeled samples, where the EC50 is inversely proportional to the affinity of the sample for the Fc gamma receptor.

Measurements of competitive binding to CD32a as determined by TR-FRET are summarized in Table 17. Increasing the number of Fc domains increased the ability of the construct to compete with immunoglobulins for CD32a, as reflected by the reduced IC50 values. Control compounds identical in sequence to S3Y-AA-OBI and S3Y-AA-AVE, but lacking the Fab domain, competed for cell surface CD32a comparable to those constructs, indicating that inclusion of the Fab domain is the Fc receptor. Demonstrate that the binding to is not altered.

[Table 17]

Example 41: Antigen Binding is Conserved in Anti-CD38 Constructs

Antigen binding was assessed using SPR. Recombinant, histidine tagged, CD38 (9049-B7, R&D Systems) protein was captured on the sensor using pre-immobilized anti-6X His antibody. Serial dilutions of the allogeneic antibody and SIF-body were passed over the sensor and the sensor was regenerated using a low pH glycine solution between analyte injections. Binding was calculated using a 1:1 Langmuir interaction model.

Binding of anti-CD38 constructs is shown in Table 18. All tested compounds were at least 93% pure by SEC. The construct had antigen binding comparable to that of the corresponding monoclonal antibody in an assay where 1:1 binding was favorable.

[Table 18]

Table 19 provides data for binding of anti-CD38 constructs in separate studies.

[Table 19]

Example 42: Anti-CD38 Fc constructs exhibit increased cytolytic activity against human lymphoma cells

As shown in Figures 31A and 31B, the S3Y-AA-CD38 anti-CD38 Fc construct is an anti-CD38 Fc construct with the same Fabs for ADCC (primary human NK cell mediated), ADCP (primary human macrophage mediated) and CDC. - more potent than the CD38 mAb.

Example 43: Anti-CD38 Fc Construct Enhances Tumor Cell Depletion from Whole Blood with Better Potency and Efficacy than Anti-CD38 Antibodies

The results of this assay are shown in Figure 32, in which human whole blood was spiked with CFSE-labeled Daudi cells and then treated with S3Y-AA-CD38, or anti-CD38 mAb with the same Fab. Changes in tumor cell population (CFSE ⁺ CD19 ⁺ ) in whole blood from baseline were determined by flow cytometry. The anti-CD38 Fc construct demonstrated 40-100 fold higher potency than the anti-anti-CD38 mAb (number of donors n=5).

Example 44: Anti-CD38 Fc constructs mediate cytotoxicity in both high CD38 complement inhibitory protein expressing tumor cell lines and low CD38 complement inhibitory protein expressing tumor cell lines

Response to CD38-targeting antibody anti-CD38 mAb correlates with CD38 expression levels on tumor cells. In addition, increased expression of complement inhibitory proteins (CD55, CD59) significantly reduced anti-CD38 mAb induced tumor cell depletion, leading to disease progression (Nijhof et al. (2016) Blood 128:959). . As shown in Figure 33, the S3Y-AA-CD38 anti-CD38 Fc construct (inverted triangle) contained Daudi cells (relatively high CD38 expression and relatively low CD55 and CD59 expression) and, importantly, Raji cells (relatively Both low CD38 expression and relatively high CD55 and CD59 expression) showed stronger CDC activity than the anti-CD38 mAb with the same Fab (circle).

Example 45: Anti-CD38 Fc constructs mediate cytotoxicity in both high CD38 complement inhibitory protein expressing tumor cell lines and low CD38 complement inhibitory protein expressing tumor cell lines

The anti-cyno CD38 Fc construct described above in Table 6, S3Y-AA-cyno, which binds to both human CD38 and cynomolgus monkey CD38, is a mAb with the same Fab, as shown in FIGS. 34A and 34B . (Anti-cyno CD38 mAb) demonstrated improved ADCC, ADCP, and CDC for human lymphoma cells.

Example 46: Anti-Cyno CD38 Fc Construct Enhances Tumor Cell Depletion from Cynomolgus Monkey Whole Blood with Better Potency and Efficacy than Anti-Cyno CD38 Antibody

The results of this assay are shown in Figure 35, in which cynomolgus monkey whole blood is spiked with CFSE-labeled Daudi cells followed by S3Y-AA-cynoCD38, or anti-CD38 mAb with the same Fab. treated with Changes in tumor cell population (CFSE ⁺ CD19 ⁺ ) in whole blood from baseline were determined by flow cytometry. The anti-cyno CD38 Fc construct demonstrated significantly higher potency than the anti-cyno CD38 mAb (n=3).

Example 47: Anti-cyno CD38 Fc construct is CD38 superior to anti-cyno CD38 mAb in cynomolgus monkeys ^high show B cell depletion

The results of this assay are shown in Figure 36, in which S3A-AA-cynovalent in vitro (left panel) as measured by B cell depletion from peripheral blood collected from cynomolgus monkeys; and superior to the anti-cyno CD38 mAb in vivo (right panel) as measured in a single-dose PD study in cynomolgus monkeys examined after 4 h of B cell depletion.

Example 48: Anti-CD38 Fc constructs show superior depletion of plasma cells from multiple myeloma patients with high myeloid plasma cell load

The results of this assay are shown in FIG. 37 , in which S3Y-AA-CD38 was superior to the anti-CD38 mAb with the same Fab sequence. Frozen bone marrow mononuclear cells (BM-MNC) from multiple myeloma patient MM536, a recurrent patient with a BM plasma cell load of 82%, were obtained from the vendor. BM-MNCs were thawed and 18 h in RPMI 1640 + 20% human serum complement in the presence or absence of varying concentrations of either anti-CD38 mAb or S3Y-AA-CD38 (to enable CDC-mediated cell death). incubated for a while. The next day, samples were stained and analyzed by FACS to assess depletion of CD138 ⁺ cells used as surrogate markers for CD38 expressing plasma/myeloma cells based on co-expression of the two markers as determined by phenotypic analysis of untreated cells. . Cell depletion was determined using viable CD138 ⁺ cell frequencies among total single cells, with all relative cell frequencies normalized to baseline frequencies observed in untreated controls (set at 0% change).

Depletion of CD138 ⁺ cells from total BM-MNCs of patient MM536 was observed after treatment with either 100 or 1000 nM of either S3Y-AA-CD38 or anti-CD38 mAbs, whereas at concentrations of 10 nM for either treatment No depletion was observed. Saturation depletion of >90% viable CD138 ⁺ cells was observed at S3Y-AA-CD38 concentrations of 100 or 1000 nM. Anti-CD38 mAb-mediated depletion was significantly lower than that observed with S3Y-AA-CD38, with a maximum depletion level of 24% at concentrations of 100 and 1000 nM, which appears to be at or near saturation. . Given the high BM plasma cell frequency (approximately 82%) in patient MM536, these results indicate that having a high bone marrow plasma cell load has been shown to have a lower objective response rate to anti-CD38 mAb treatment in clinical applications. In MM patients, the use of anti-CD38 Fc constructs may show the potential for greater response.

Example 48: Anti-CD38 Fc constructs show enhanced binding to cell surface FcγR and human serum complement

38A shows the results of a study showing that S3Y-AA-CD38 binding to FcgRIIa, FcgRIIIa and complement is at least 100-fold greater than that of an anti-CD38 mAb.

Figure 38b shows the results of a study showing that S3Y-AA-CD38 showed greater than 500-fold enhanced binding to FcγRIIa, FcγRIIIa on the immune cell surface, and 12-fold enhanced Clq complement protein binding than anti-CD38 mAb.

Example 49: Anti-CD38 Fc constructs with mutant Fc CH2 domains show longer lasting B cell depletion

Similar to Example 38, cyno whole blood was mixed with serial dilutions of each of anti-cyno CD38 mAb, anti-cyno CD38 construct 13 (S3Y-AA-cyno-001), and B cell depletion was measured. did. In addition, the anti-cyno CD 13 construct was tested as a variant. Each CH2 domain had an R292P mutation (see FIGS. 24A and 24B for Fc domain numbering). Thus, this construct had the aforementioned Fc domain mutations. Table 20 below provides the amino acid sequences of the polypeptides constituting this variant CD38 construct 13 variant (mutations are in bold, highlighted and underlined). S3Y-CD38 (CC R292P) is a version that binds to human CD38, and S3Y-cyno-001 (CC R292P) binds to cynomolgus monkey CD38. As can be seen in Figure 39, S3Y-cyno-001 (CC R292P) (“second generation SIF body”) is combined with S3Y-cyno-001 (without R292P mutation) (“first generation SIF body”) and In contrast, it showed superior sustainability of B cell depletion.

[Table 20]

Example 50: Binding of CD38 mAb and SIF body to CD38

CD38 binding to mAbs and SIF-bodies was measured using a Biacore 8K instrument (GE Healthcare). Protein A immobilized on a CM5 sensor chip was used to capture the test molecule (SIF-body or antibody) from solution. Serial dilutions of CD38 were flowed over the captured test molecules. This assay was run as a single-cycle kinetics assay, in which increasing concentrations of CD38 are injected over the captured test molecule and dissociation is monitored at the end point. Sensorgrams were double-reference subtracted and fitted to a 1:1 binding model. The kinetic association constant (ka) and the kinetic dissociation constant (kd) were measured, and the equilibrium dissociation constant (KD) was calculated by dividing kd by ka. The stoichiometry of binding to the test molecule was also determined. The capture level of each test molecule, measured in response units (RU), was multiplied by the ratio of CD38 to the molecular weight of the test molecule to obtain the theoretical maximum level of binding. The stoichiometry was obtained by dividing the RMax value for this experiment by the theoretical maximum binding level. Cynomolgus monkey CD38 binding was tested by measuring the binding of 400 nM and 200 nM protein to each test molecule.

Figure 41 shows the results of analysis of human CD38 binding to cyno anti-CD38 antibody and cyno SIF-body as measured by SPR. The binding sensorgram and 1:1 binding model fit for the anti-CD38 molecule are shown on the left. Kinetic constants and equilibrium constants are shown in the middle and upper right. The stoichiometry of human CD38 binding to an anti-CD38 molecule is shown in the lower right. All constructs bind human CD38 equally. S3Y-CC-CD38 is construct 13 with an R292P mutation in the Fc CH2 mutation, and S3Y-AA-CD38 lacks the R292P mutation but is otherwise identical.

Figure 42 shows the results of analysis of human CD38 binding to cyno-CD38 antibody and cyno-SIF-body measured by SPR. Binding sensorgrams and 1:1 binding model fit for the cyno-CD38 molecule are shown on the left. Kinetic constants and equilibrium constants are shown in the middle and upper right. The stoichiometry of human CD38 binding to the cyno-CD38 molecule is shown in the lower right. All constructs bind human CD38 equally.

Figure 43 shows the results of analysis of sinenomolgus CD38 binding to cyno-CD38 antibody, cyno-CD38 SIF-body and CD38 mAb as measured by SPR. Binding sensorgrams and 1:1 binding model fit for the cyno-CD38 molecule are shown in the left panel and bottom middle panel. Binding sensorgrams and 1:1 binding model fit for CD38 mAb are shown in the upper middle panel. Equilibrium constants are shown in the right panel. All cyno-CD38 constructs bind equally to cynomolgus CD38. CD38 mAb does not bind cynomolgus CD38.

Example 51: Binding of S3Y-AA-CD38 and S3Y-CC-CD38 to human FcγR

44 shows the results of analysis of binding of S3Y-AA-CD38 and S3Y-CC-CD38 to human FcγR versus CD38 mAb using an Fc-gamma receptor homogeneous time resolved fluorescence (HTRF) assay. of S3Y-AA-CD38, S3Y-CC-CD38 and CD38 mAbs in humans against FCGR1A, FCGR2A (H167), FCGR2A (R167), FCGR2B, FCGR3A (F176) and FCGR3A (V176) using a competitive HTRF assay. binding was determined. IC ₅₀ inhibition constants were derived from this data. Binding of S3Y-AA-CD38 and S3Y-CC-CD38 is expressed as fold change in IC50 versus CD38 MAb IC50. S3Y SIF exhibits strongly enhanced binding to cell surface receptors. The R292P mutation in S3Y-CC-CD38 results in a greater than 100-fold reduction in binding to FcgR2b compared to the parental S3Y-AA-CD38 molecule.

The Fc-gamma receptor competitive HTRF assay was purchased from Cis-Bio and performed according to the manufacturer's instructions. Assays were performed for FCGR1A, FCGR2A (H167), FCGR2A (R167), FCGR2B, FCGR3A (F176) and FCGR3A (V176). Fluorescence measurements were performed at 620 nm and 655 nm using a BMG Pharastar fluorometer. A ratio of 655/620*10000 was calculated for each well. The data was copied into GraphPad Prism for analysis. Concentration values were log-transformed and the data fit to a 4-parameter curve limiting the top and bottom values to be common among all tested samples. IC50 values were estimated and reported for each independent dilution.

Example 52: Binding of anti-CD38 mAb and S3Y-CC-CD38 molecule to endogenous CD38 receptor on human tumor cells.

CD38 is a cell surface type II glycoprotein receptor with a large extracellular region and a very short cytosolic domain. This receptor is expressed in a number of hematologic malignancies derived from both B cell and plasma cell lineages, including multiple myeloma (MM). Cell lines derived from lymphoma and myeloma patients were selected for CD38 expression analysis. CD38 levels were determined by binding of fluorescently labeled anti-CD38 mAbs by flow cytometry. Cells were incubated with VivoTag 645-labeled anti-CD38 mAb at 4° C. for 30 min. Cells were then washed, fixed, and single cell events were acquired on a Verse flow cytometer. Values represent mean ± SD. The hierarchy of cell surface CD38 expression is Daudi cells (lymphoma cell line) > Raji (lymphoma cell line) > SU-DHL (lymphoma cell line) > MM.1S (myeloma cell line) > RPMI8226 (myeloma cell line) ( FIG. 45 ).

Daudi and Raji cell lines with high and moderate levels of cell surface CD38 were selected for cell-based functional assays. After a dose-dependent increase in binding, there was saturation with anti-CD38 mAb and S3Y molecules in both the Daudi and Raji cell lines ( FIG. 46 ). The binding profiles of the anti-CD38 mAbs, S3Y-CC-CD38, and S3Y-AA-CD38 were similar, indicating the effect of mutations in the Fc domain of S3Y-CC-CD38 on the interaction of these molecules with endogenous cell surface CD38. suggests that there was no Cells were incubated with VivoTag 645-labeled anti-CD38 mAb or S3Y-AA or S3Y-CC molecules at 4° C. for 30 min. Cells were then washed, fixed, and single cell events were acquired on a Cytek Aurora flow cytometer. Values represent mean ± SD. As shown in Figure 46, S3Y-CC-CD38 and anti-CD38 mAbs show similar binding to lymphoma cell surface CD38.

Example 53: Effect of S3Y-CC-CD38 and anti-CD38 mAbs on Fc effector function based cell depletion assay

Therapeutic anti-CD38 antibody daratumumab (Darzalex) provides Fc-dependent immunity, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). It kills tumor cells through an effector mechanism. CDC, ADCC, and ADCP assays were performed to assess the relative cytotoxicity of S3Y-CC-CD38 to the maternal version S3Y-AA-CD38 and anti-CD38 mAbs.

To evaluate CDC activity, Daudi or Raji cells were incubated with human serum complement and drug molecules at 37° C. for 2 h to promote complement-mediated lysis. Then, Alamar Blue (cell viability reagent) was added to each well, incubated at 37° C. for 18 hours, and viable cell fluorescence was measured using a fluorometer to determine the degree of cell lysis. Both S3Y and anti-CD38 mAbs showed a dose-dependent increase in complement-mediated Daudi cell lysis ( FIG. 47A , left panel). Raji cells have lower CD38 expression than Daudi cells, but they also have higher levels of complement inhibitory proteins on their cell surface. Although anti-CD38 mAb failed to induce CDC against Raji cells, the S3Y molecule showed a clear dose-dependent cytotoxicity ( FIG. 47B , right panel). Overall, S3Y-CC-CD38 showed better potency and maximal target cell lysis in cells where the anti-CD38 mAb failed to induce lysis by CDC.

Example 54: CD38 ⁺ Enhanced potency and efficacy of S3Y-CC-CD38A in inducing cytolytic activity against human lymphoma cells.

ADCC and ADCP were dependent on the effective interaction between the Fc region of the antibody and the Fcγ receptor (FcyR) expressed on immune effector cells. During induction of ADCC, lysis of antibody coated tumor cells by effector cells occurs. NK cells play an important role in ADCC mediated by therapeutic antibodies. Importantly, CD38-targeting S3Y molecules are distinguished from anti-CD38 mAbs by their ability to bind with much stronger affinity due to their vigorous binding to FcγR. The relative ADCC activities of S3Y-CC-CD38, S3Y-AA-CD38, and anti-CD38 mAbs were evaluated. For this assay, primary human NK cells were added to the tumor cells. Then, after the cell mixture was incubated with any one of the drug molecules at 37° C. for 5 hours, dead cells were detected by CytoTox Glo reagent. The S3Y molecule clearly shows a significantly enhanced potency over the reference anti-CD38 mAb ( FIG. 48A ).

In the course of ADCP, phagocytosis of antibody-opsonized tumor cells occurs through the binding of FcγRs (eg, FcγRIIA and FcγRIIIA) present on monocytes and macrophages. Phagocytosis contributes to the anti-tumor activity of CD38-targeting antibodies. Interestingly, individual macrophages have the ability to rapidly and sequentially completely encapsulate large numbers of daratumumab-coated tumor cells, indicating that ADCP is an efficient killing mechanism of daratumumab (Darzalex). We selected the M2c phenotype of macrophages as effector cells because they are more prevalent in the bone marrow microenvironment in hematologic malignancies. To determine the relative ADCP activity of the S3Y molecule and the reference anti-CD38 mAb, monocytes purified from human PBMC were cultured in M-CSF, followed by incubation with IL-10 to generate M2c macrophages. Phagocytosis of tumor cells by macrophages was captured by real-time imaging. S3Y-CC-CD38 and S3Y-AA-CD38 clearly show significantly enhanced potency over the reference anti-CD38 mAb in inducing ADCP ( FIG. 48B ).

Example 55: S3Y-CC-CD38 enhances lymphoma cell depletion from whole blood with better potency and cytotoxicity.

The relative cytotoxic activity of the S3Y molecule relative to the anti-CD38 mAb was further compared in a whole blood tumor cell (CD38-high Daudi cells, and moderate-CD38 expressing Raji cells) depletion assay, which incorporates different antibody modes of action. (CDC, ADCC, and induction of cell death). Daudi or Raji lymphoma cells labeled with CFSE dye were spiked into heparinized blood samples from healthy volunteers and in the presence of anti-CD38 mAb or S3Y-CC-CD38 or S3Y-AA-CD38 at 37°C for 18 hours. incubated. Blood cells were then stained for surface markers, fixed and acquired on a flow cytometer. A decrease in the frequency of CFSE ⁺ (Daudi or Raji) cells in the presence of anti-CD38 mAb or S3Y-molecule versus no drug treatment was indicative of selective cell depletion. The superiority of S3Y-CC-CD38 compared to anti-CD38 mAb is indicated by higher potency values and by higher maximal CFSE ⁺ Daudi or Raji cell depletion in whole blood ( FIGS. 49A and 49B ).

Example 56: S3Y-CC-CD38 and S3Y-AA-CD38 in vitro ( ex-vivo ) mediates enhanced cytotoxicity against plasma cells in bone marrow biopsies from multiple myeloma patients in a native environment assay.

The therapeutic anti-CD38 mAb Darzalex was approved by the FDA based on a monotherapy trial with a 31% clinical response rate in a population of patients with relapsed and refractory multiple myeloma (RRMM) with more than 4 prior therapies. Today, Darzalex is commonly used in combination with iMIDs and proteasome inhibitors in frontline and relapsed and refractory settings for patients with MM. In these combo trials, achieving a higher minimal residual disease (MRD) status based on flow cytometry is considered as one of the key predictors of better progression-free survival in patients. Patients are considered MRD-negative if they have less than 1 plasma cell (CD138 ⁺ )/100,000 bone marrow cells when tested by a validated clinical flow cytometry assay. Retrospective data from the Darzalex combo trial (POLLUX study) indicate that a significant number of patients do not achieve an MRD-negative status. Therefore, a better plasma cell depleting agent may achieve a better duration of disease-free remission than the current SOC in RRMM patients.

The plasma cell depleting activity of S3Y-CC-CD38 and its parent molecule S3Y-AA-CD38 compared to daratumumab (Darzalex) was investigated in an ex vivo assay using bone marrow aspirates from multiple myeloma (MM) patients. Bone marrow aspirates were collected from patients with relapsed and refractory MM (n=5) with prior therapy including corticosteroids, stem cell transplantation, proteasome inhibitor (Velcade), iMID (lenalidomide). All these patients had varying levels of plasma cell load in their bone marrow as determined by a panel of plasma cell surface markers using flow cytometry. An ex vivo plasma cell depletion assay was performed using fresh bone marrow aspirate ( FIG. 50 ). Bone marrow samples were extracted into Vacutainer tubes with heparin as anticoagulant and further diluted using IMDM supplemented with 20% (v/v) autologous plasma to maintain the native environment. The mixture was dispensed into 96-well plates containing varying concentrations of either daratumumab (Darzalex) or S3Y-CC-CD38 or its parent molecule S3Y-AA-CD38. The plates with the samples were then incubated for 3 hours at 37° C. in humidified air containing 5% CO ₂ . To prepare samples for flow cytometry analysis, at the end of incubation, RBCs were lysed and then single fluorescently-labeled for cell surface markers (CD38, CD16, CD319, CD45, CD56, CD3) and Annexin V. Viable plasma cells remaining in each well were identified and quantified by staining with a panel of clonal antibodies.

As expected, Darzalex depleted plasma cells from bone marrow in a dose-dependent manner ( FIG. 50 ). Both S3Y molecules (S3Y-CC-CD38 and S3Y-AA-CD38) showed a superior dose-dependent depletion of multiple myeloma tumor cells in all patients. S3Y-CC-CD38 and S3Y-AA-CD38 showed better efficacy than Darzalex (FIG. 50). In addition, in 2 of 5 patient bone marrow samples, the S3Y molecule abolished the majority of plasma cells (>95%). Overall, data from these 5 patients suggest that the S3Y-CC-CD38 and S3Y-AA-CD38 molecules are superior to Darzalex in depleting MM tumor cells from bone marrow aspirate ( FIG. 50 ).

Example 57: S3Y-CC-cyno-CD38, S3Y-AA-cyno-CD38, and anti-cyno-CD38 mAbs show similar binding to cell surface CD38 on human lymphoma cell lines.

A mutation (R292P) was introduced in the Fc domain of the S3Y-AA-cyno-CD38 molecule to generate S3Y-CC-cyno-CD38. Fc mutations reduce its binding to Fc gamma RIIB (see FIG. 45 ). Functional activity was confirmed in cell-based assays. The PK-optimized molecule was then tested in cynomolgus monkeys to determine if Fc mutations enhance the depth and duration of the PD response and enhance the serum half-life of the S3Y-CC-cyno-CD38 molecule.

The S3Y-CC-cyno-CD38, S3Y-AA-cyno-CD38, and anti-cyno-CD38 mAbs bind to both human and cynomolgus monkey CD38 ( FIG. 51 ). To confirm that the R292P Fc mutation in the S3Y-CC-cyno-CD38 molecule has no effect on its ability to bind cell surface CD38, its parent molecule of labeled S3Y-CC-cyno-CD38 (S3Y -AA-cyno-CD38) and to the reference anti-cyno-CD38 mAb were compared in Daudi and Raji cells. S3Y-CC-cyno-CD38 shows similar binding and EC50 values as the parental S3Y-AA-cyno-CD38 and reference anti-cyno-CD38 mAbs.

Example 58: S3Y-CC-cyno-CD38 showed enhancement of Fc effector function-mediated target cell cytotoxicity over anti-cyno-CD38 mAb.

Both the S3Y-CC-cyno-CD38 and reference anti-cyno-CD38 mAbs bind human and cyno CD38. Fc effector function was evaluated based on the cell depleting activity by this S3Y molecule with an Fc mutation. Many Fc mutations affect complement binding to antibody-opsonized cell surfaces, and also interactions with Fc gamma receptors on NK cells and macrophages. Therefore, Daudi and Raji cells were used as target cell lines. The effect of Fc mutations in S3Y-CC-cyno-CD38 on cell death activity in CDC, ADCC, and ADCP assays was investigated as previously described. S3Y-CC-cyno-CD38 showed significantly superior potency in inducing cellular cytotoxicity in all Fc effector function-based assays using human complement, primary human NK cells, and M2c macrophages ( Fig. 52).

Example 59: Exploratory PK/PD and Tolerability Study in Cynomolgus Monkeys with S3Y-CC-Cyno-CD38

Pharmacokinetic properties (PK), tolerability, and sustainability of pharmacodynamic effects (B cell depletion) of S3Y-CC-cyno-CD38 (with R292P mutation in Fc domain) by performing a single dose study in cynomolgus monkeys. ) was evaluated. Superiority to and differentiation from the parent molecule S3Y-AA-cyno-CD38 and the reference anti-cyno-CD38 mAb was also investigated. To this end, the 15-day single-dose pharmacokinetics, pharmacodynamics of S3Y-CC-cyno-CD38, S3Y-AA-cyno-CD38, and anti-cyno-CD38 mAbs after intravenous infusion in cynomolgus monkeys. Clinical characteristics, and tolerability studies were performed. Exposure at steady state (area under the serum concentration-time curve from dose administration to 360 hours post-dose) generally increased in a dose-proportional manner ( FIG. 53 ). S3Y-CC-cyno-CD38 showed a significantly improved PK profile with higher serum drug concentration from its parent molecule S3Y-AA-cyno-CD38 ( FIG. 53 ).

Peripheral of S3Y-CC-cyno-CD38, S3Y-AA-cyno-CD38, and anti-cyno-CD38 mAb-treated animals using antibodies against CD138, CD159a, CD27, CD20, CD19, and CD3 Cell immunophenotyping was performed on blood. Flow cytometry was used to determine an estimate of the relative percentage of positive cells and used in the calculation of absolute cell counts. Absolute cell counts at each time point are normalized to pre-dose levels for individual animals. Results discussed herein represent % change in B cell counts in peripheral blood normalized to baseline (set at 100%) prior to dosing. Flow cytometry analysis showed that administration of the drug molecule resulted in a mild to significant decrease in absolute cell counts (total B cells) of CD19 ⁺ CD20 ^+/- in all treatment groups ( FIG. 54 ). S3Y-CC-cyno-CD38 (1.7 mg/Kg) is anti-between its parent molecule S3Y-AA-cyno-CD38 (1.7 mg/Kg) and in terms of its pharmacodynamic effect on B cells at equimolar dose. showed much higher potency and efficacy than the no-CD38 mAb (1.0 mg/Kg) ( FIG. 54 ).

Example 60: Multiple Repeat-Dose Study: All S3Y-CC-cyno-CD38 doses were potent and efficacious in the repeat dose setting; Plasma cells (CD138 ⁺ ) represents only a subpopulation of all lymphoid cells. S3Y-CC-cyno-CD38 can deplete plasma cells from tissues in a dose-dependent manner.

The pharmacodynamic effects of S3Y-CC-cyno-CD38 were investigated in a 4-week repeat-dose (once weekly for 4 weeks) study in cynomolgus monkeys. For this study, a 10-fold higher dose (1.7, 17, 51 mg/Kg) for S3Y-CC-cyno-CD38 was used compared to the initial study ( FIGS. 53 and 54 ) to achieve maximum tolerability, safety as well as In addition, multiple PD efficacy readings, cell depletion in blood, and plasma cell depletion from lymphoid tissues were evaluated. Blood samples were collected during this study for hematological evaluation, flow cytometry, and tissues harvested after completion of administration to evaluate the effect of treatment on plasma cell depletion. All treatment groups showed good tolerability. S3Y-CC-CD38B treatment depleted circulating B cells in a dose-dependent manner with improved sustainability of effect with increasing dose ( FIG. 55A ). Bone marrow and spleen represent the major lymphoid tissues. Plasma cells (CD138 ⁺ ) represent only a subpopulation of all lymphoid cells ( FIG. 55B ). S3Y-CC-cyno-CD38 depleted plasma cells from bone marrow and spleen in a dose-dependent manner ( FIG. 55B ).

S3Y-CC-cyno-CD38 is a novel protein structure, for which there is no evidence for the feasibility of subcutaneous (SC) administration. The increased molecular weight of this molecule may lead to interference with absorption via the SC pathway. The CD38 therapeutic is commercially available in both IV and SC configurations, and S3Y-CC-cyno-CD38 will preferably be available in both configurations. Non-GLP single IV and SC doses of S3Y-CC-cyno-CD38 in cynomolgus monkeys Pharmacokinetic (PK), pharmacodynamic (PD) and tolerability evaluations were performed. This study showed no tolerability findings, and the PK and PD results showed the feasibility of subcutaneous delivery of the S3Y-CC-cyno-CD38 molecule in non-human primates. The data also suggest relatively better bioavailability and PD effects with high-dose SC injections ( FIGS. 56A , 56B ).

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the present invention has been described with respect to specific embodiments thereof, further modifications are possible and the present application generally follows the principles of the present invention and falls within known or customary practice in the art, It will be understood that the intention is to cover any modification, use, or adaptation of the invention, including such departures from the invention, which may apply to the essential features described above and fall within the scope of the invention.

Other embodiments are within the scope of the claims.

SEQUENCE LISTING <110> MOMENTA PHARMACEUTICALS, INC. <120> COMPOSITIONS AND METHODS RELATED TO ENGINEERED FC-ANTIGEN BINDING DOMAIN CONSTRUCTS TARGETED TO CD38 <130> 14131-0219WO1 <140> PCT/US2020/051663 <141> 2020-09-18 <150> 62/902,380 <151> 2019-09-18 <160> 250 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 1 Gly Gly Gly Gly Ser 1 5 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 2 Gly Gly Ser Gly One <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Ser Gly Gly Gly One <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Gly Ser Gly Ser One <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Gly Ser Gly Ser Gly Ser 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Gly Ser Gly Ser Gly Ser Gly Ser 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Gly Gly Ser Gly Gly Ser 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Gly Gly Ser Gly Gly Gly Ser Gly 1 5 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Ser Gly 20 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly 20 <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Gly Gly Gly Gly One <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 <210> 23 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly 20 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Gly Gly Gly Gly Gly 1 5 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 <210> 26 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 <210> 27 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly 20 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly 1 5 10 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ser Ala Cys Tyr Cys Glu Leu Ser 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Arg Ser Ile Ala Thr 1 5 <210> 31 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Arg Pro Ala Cys Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn 1 5 10 15 His <210> 32 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser 1 5 10 15 Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly 20 25 30 Thr Gly Ser Gly 35 <210> 33 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Ala Ala Ala Asn Ser Ser Ile Asp Leu Ile Ser Val Pro Val Asp Ser 1 5 10 15 Arg <210> 34 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 1 5 10 15 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser 35 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 36 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 Gly Ser <210> 37 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 <210> 38 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 38 His His His His His His 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 42 <211> 227 <212> PRT <213> Homo sapiens <400> 42 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 43 <211> 232 <212> PRT <213> Homo sapiens <400> 43 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 44 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 45 <211> 226 <212> PRT <213> Homo sapiens <400> 45 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 46 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 47 <211> 231 <212> PRT <213> Homo sapiens <400> 47 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly 225 230 <210> 48 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 48 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 49 <400> 49 000 <210> 50 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 50 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 51 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 52 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 53 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 54 <400> 54 000 <210> 55 <400> 55 000 <210> 56 <400> 56 000 <210> 57 <400> 57 000 <210> 58 <400> 58 000 <210> 59 <400> 59 000 <210> 60 <400> 60 000 <210> 61 <400> 61 000 <210> 62 <400> 62 000 <210> 63 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 63 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 64 <400> 64 000 <210> 65 <400> 65 000 <210> 66 <400> 66 000 <210> 67 <400> 67 000 <210> 68 <400> 68 000 <210> 69 <400> 69 000 <210> 70 <400> 70 000 <210> 71 <211> 945 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp 595 600 605 Lys Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly 690 695 700 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp 705 710 715 720 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 725 730 735 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 740 745 750 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 755 760 765 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 770 775 780 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 785 790 795 800 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 805 810 815 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 820 825 830 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 835 840 845 Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu 850 855 860 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 865 870 875 880 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 885 890 895 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 900 905 910 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 915 920 925 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 930 935 940 Gly 945 <210> 72 <400> 72 000 <210> 73 <211> 945 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 73 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp 595 600 605 Lys Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly 690 695 700 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp 705 710 715 720 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 725 730 735 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 740 745 750 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 755 760 765 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 770 775 780 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 785 790 795 800 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 805 810 815 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 820 825 830 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 835 840 845 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 850 855 860 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 865 870 875 880 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 885 890 895 Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp 900 905 910 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 915 920 925 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 930 935 940 Gly 945 <210> 74 <400> 74 000 <210> 75 <400> 75 000 <210> 76 <400> 76 000 <210> 77 <400> 77 000 <210> 78 <400> 78 000 <210> 79 <400> 79 000 <210> 80 <400> 80 000 <210> 81 <400> 81 000 <210> 82 <400> 82 000 <210> 83 <400> 83 000 <210> 84 <400> 84 000 <210> 85 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Gly Phe Thr Phe Asn Ser Phe Ala 1 5 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Gly Tyr Thr Phe Thr Asp Tyr Trp 1 5 <210> 87 <400> 87 000 <210> 88 <400> 88 000 <210> 89 <400> 89 000 <210> 90 <400> 90 000 <210> 91 <400> 91 000 <210> 92 <400> 92 000 <210> 93 <400> 93 000 <210> 94 <400> 94 000 <210> 95 <400> 95 000 <210> 96 <400> 96 000 <210> 97 <400> 97 000 <210> 98 <400> 98 000 <210> 99 <400> 99 000 <210> 100 <400> 100 000 <210> 101 <400> 101 000 <210> 102 <400> 102 000 <210> 103 <400> 103 000 <210> 104 <400> 104 000 <210> 105 <400> 105 000 <210> 106 <400> 106 000 <210> 107 <400> 107 000 <210> 108 <400> 108 000 <210> 109 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Ile Tyr Pro Gly Asp Gly Asp Thr 1 5 <210> 110 <400> 110 000 <210> 111 <400> 111 000 <210> 112 <400> 112 000 <210> 113 <400> 113 000 <210> 114 <400> 114 000 <210> 115 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 115 Ile Ser Gly Ser Gly Gly Gly Thr 1 5 <210> 116 <400> 116 000 <210> 117 <400> 117 000 <210> 118 <400> 118 000 <210> 119 <400> 119 000 <210> 120 <400> 120 000 <210> 121 <400> 121 000 <210> 122 <400> 122 000 <210> 123 <400> 123 000 <210> 124 <400> 124 000 <210> 125 <400> 125 000 <210> 126 <400> 126 000 <210> 127 <400> 127 000 <210> 128 <400> 128 000 <210> 129 <400> 129 000 <210> 130 <400> 130 000 <210> 131 <400> 131 000 <210> 132 <400> 132 000 <210> 133 <400> 133 000 <210> 134 <400> 134 000 <210> 135 <400> 135 000 <210> 136 <400> 136 000 <210> 137 <400> 137 000 <210> 138 <400> 138 000 <210> 139 <400> 139 000 <210> 140 <400> 140 000 <210> 141 <400> 141 000 <210> 142 <400> 142 000 <210> 143 <400> 143 000 <210> 144 <400> 144 000 <210> 145 <400> 145 000 <210> 146 <400> 146 000 <210> 147 <400> 147 000 <210> 148 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 148 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr 1 5 10 15 <210> 149 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 149 Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr 1 5 10 <210> 150 <400> 150 000 <210> 151 <400> 151 000 <210> 152 <400> 152 000 <210> 153 <400> 153 000 <210> 154 <400> 154 000 <210> 155 <400> 155 000 <210> 156 <400> 156 000 <210> 157 <400> 157 000 <210> 158 <400> 158 000 <210> 159 <400> 159 000 <210> 160 <400> 160 000 <210> 161 <400> 161 000 <210> 162 <400> 162 000 <210> 163 <400> 163 000 <210> 164 <400> 164 000 <210> 165 <400> 165 000 <210> 166 <400> 166 000 <210> 167 <400> 167 000 <210> 168 <400> 168 000 <210> 169 <400> 169 000 <210> 170 <400> 170 000 <210> 171 <400> 171 000 <210> 172 <400> 172 000 <210> 173 <400> 173 000 <210> 174 <400> 174 000 <210> 175 <400> 175 000 <210> 176 <400> 176 000 <210> 177 <400> 177 000 <210> 178 <400> 178 000 <210> 179 <400> 179 000 <210> 180 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 180 Gln Ser Val Ser Ser Tyr 1 5 <210> 181 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 181 Gln Asp Val Ser Thr Val 1 5 <210> 182 <400> 182 000 <210> 183 <400> 183 000 <210> 184 <400> 184 000 <210> 185 <400> 185 000 <210> 186 <400> 186 000 <210> 187 <400> 187 000 <210> 188 <400> 188 000 <210> 189 <400> 189 000 <210> 190 <400> 190 000 <210> 191 <400> 191 000 <210> 192 <400> 192 000 <210> 193 <400> 193 000 <210> 194 <400> 194 000 <210> 195 <400> 195 000 <210> 196 <400> 196 000 <210> 197 <400> 197 000 <210> 198 <400> 198 000 <210> 199 <400> 199 000 <210> 200 <400> 200 000 <210> 201 <400> 201 000 <210> 202 <400> 202 000 <210> 203 <400> 203 000 <210> 204 <400> 204 000 <210> 205 <400> 205 000 <210> 206 <400> 206 000 <210> 207 <400> 207 000 <210> 208 <400> 208 000 <210> 209 <400> 209 000 <210> 210 <400> 210 000 <210> 211 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 211 Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5 <210> 212 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 212 Gln Gln His Tyr Ser Pro Pro Tyr Thr 1 5 <210> 213 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 213 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 20 25 30 <210> 214 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(30) <223> This sequence may encompass 4-30 residues <400> 214 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 <210> 215 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(30) <223> This sequence may encompass 8-30 residues <400> 215 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 <210> 216 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (1)..(30) <223> This sequence may encompass 12-30 residues <400> 216 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 <210> 217 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(20) <223> This sequence may encompass 4-20 residues <400> 217 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly 20 <210> 218 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(20) <223> This sequence may encompass 8-20 residues <400> 218 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly 20 <210> 219 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(20) <223> This sequence may encompass 12-20 residues <400> 219 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly 20 <210> 220 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu 20 <210> 221 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 221 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 <210> 222 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 222 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu <210> 223 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 223 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1 5 10 15 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 20 25 30 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 35 40 45 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50 55 60 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 65 70 75 80 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 85 90 95 Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 <210> 224 <211> 106 <212> PRT <213> Homo sapiens <400> 224 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100 105 <210> 225 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 225 Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys 20 <210> 226 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 226 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 20 25 30 <210> 227 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 227 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp 1 5 10 15 Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 <210> 228 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 228 Ser Tyr Tyr Met Asn 1 5 <210> 229 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 229 Gly Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 230 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 230 Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr 1 5 10 <210> 231 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 231 Ser Gly Asp Asn Leu Arg His Tyr Tyr Val Tyr 1 5 10 <210> 232 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 232 Gly Asp Ser Lys Arg Pro Ser 1 5 <210> 233 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 233 Gln Thr Tyr Thr Gly Gly Ala Ser 1 5 <210> 234 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 234 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr 65 70 75 80 Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 235 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 235 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val 115 <210> 236 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 236 Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly 1 5 10 15 Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 237 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 237 Asp Ile Glu Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Thr Gly Gly Ala Ser Leu 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 <210> 238 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 238 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 239 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 239 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met 20 25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp 35 40 45 Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val Lys Gly 50 55 60 Gln Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 465 470 475 480 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 485 490 495 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 500 505 510 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 515 520 525 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 530 535 540 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 545 550 555 560 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 565 570 575 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 580 585 590 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys 595 600 605 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 610 615 620 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 625 630 635 640 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 645 650 655 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 660 665 670 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 675 680 685 Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 240 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 240 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 241 <211> 698 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 241 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 595 600 605 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 242 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 242 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met 20 25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp 35 40 45 Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val Lys Gly 50 55 60 Gln Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 465 470 475 480 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 485 490 495 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 500 505 510 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 515 520 525 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 530 535 540 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 545 550 555 560 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 565 570 575 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 580 585 590 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 595 600 605 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 610 615 620 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 625 630 635 640 Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe 645 650 655 Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 660 665 670 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 675 680 685 Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 243 <211> 720 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 243 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 225 230 235 240 Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys 370 375 380 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly 465 470 475 480 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys 485 490 495 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 500 505 510 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 515 520 525 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 530 535 540 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 545 550 555 560 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 565 570 575 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 580 585 590 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 595 600 605 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 610 615 620 Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu Trp 625 630 635 640 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 645 650 655 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 660 665 670 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 675 680 685 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 690 695 700 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 705 710 715 720 <210> 244 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 244 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 245 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 245 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 225 230 235 240 Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys 370 375 380 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 <210> 246 <211> 698 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 246 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp 595 600 605 Lys Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 247 <211> 945 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 247 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 595 600 605 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly 690 695 700 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp 705 710 715 720 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 725 730 735 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 740 745 750 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 755 760 765 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 770 775 780 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 785 790 795 800 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 805 810 815 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 820 825 830 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 835 840 845 Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu 850 855 860 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 865 870 875 880 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 885 890 895 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 900 905 910 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 915 920 925 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 930 935 940 Gly 945 <210> 248 <211> 698 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 248 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 595 600 605 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 249 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 249 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met 20 25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp 35 40 45 Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val Lys Gly 50 55 60 Gln Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 450 455 460 Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys 465 470 475 480 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 485 490 495 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 500 505 510 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 515 520 525 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu 530 535 540 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 545 550 555 560 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 565 570 575 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 580 585 590 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 595 600 605 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 610 615 620 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 625 630 635 640 Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe 645 650 655 Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 660 665 670 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 675 680 685 Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 250 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 250 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225

Claims (26)

An Fc-antigen binding domain construct comprising:
a)
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a first CD38 heavy chain binding domain, and
iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;
b)
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer,
iii) a second CD38 heavy chain binding domain, and
iv) a second polypeptide comprising a linker connecting the third Fc domain monomer and the fourth Fc domain monomer;
c) a third polypeptide comprising a fifth Fc domain monomer;
d) a fourth polypeptide comprising a sixth Fc domain monomer;
e) a fifth polypeptide comprising a first CD38 light chain binding domain; and
f) a sixth polypeptide comprising a second CD38 light chain binding domain;
The first Fc domain monomer and the third Fc domain monomer together form a first Fc domain, the second Fc domain monomer and the fifth Fc domain monomer together form a second Fc domain, and the fourth Fc monomer and the sixth Fc monomer together form a third Fc domain, wherein the first CD38 heavy chain binding domain and the first CD38 light chain binding domain together form a first Fab; wherein the second CD38 heavy chain binding domain and the second CD38 light chain binding domain together form a second Fab. The Fc antigen domain construct of claim 1 , wherein the first and second polypeptides are identical in sequence. The Fc antigen domain construct of claim 1 , wherein the third and fourth polypeptides are identical in sequence. The Fc antigen domain construct of claim 1 , wherein the fifth and sixth polypeptides are identical in sequence. The method of claim 1 , wherein the first and second polypeptides are at least 95% identical to SEQ ID NO: B, the third and fourth polypeptides are at least 95% identical to SEQ ID NO: C, and the fifth and fourth polypeptides are at least 95% identical to SEQ ID NO: 6 The Fc antigen domain construct, wherein the polypeptide is at least 95% identical to SEQ ID NO: A. The method of claim 1 , wherein the first and second polypeptides are at least 98% identical to SEQ ID NO: B, the third and fourth polypeptides are at least 98% identical to SEQ ID NO: C, and the fifth and fourth polypeptides are at least 98% identical to SEQ ID NO: C. 6 The Fc antigen domain construct is at least 98% identical to SEQ ID NO: A. The method of claim 1 , wherein the first and second polypeptides comprise or consist of SEQ ID NO: B, the third and fourth polypeptides comprise or consist of SEQ ID NO: C, and the fifth and second polypeptides comprise or consist of SEQ ID NO: 6 An Fc antigen domain construct comprising or consisting of SEQ ID NO: A. 8. The method according to any one of claims 1 to 7, wherein the first and second polypeptides are identical in sequence, the third and fourth polypeptides are identical in sequence, and the fifth and sixth polypeptides are identical in sequence. are identical in sequence, the Fc antigen domain construct. As a composition,
a)
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a first CD38 heavy chain binding domain, and
iv) a first polypeptide comprising a linker connecting the first Fc domain monomer and the second Fc domain monomer;
b) a second polypeptide comprising a third Fc domain monomer; and
c) a third polypeptide comprising a CD38 light chain binding domain. 10. The method of claim 9, wherein the first polypeptide is at least 95% identical to SEQ ID NO: B, the second polypeptide is at least 95% identical to SEQ ID NO: C, and the third polypeptide is at least 95% identical to SEQ ID NO: A; composition. 10. The method of claim 9, wherein the first polypeptide is at least 98% identical to SEQ ID NO: B, the second polypeptide is at least 98% identical to SEQ ID NO: C, and the third polypeptide is at least 98% identical to SEQ ID NO: A; composition. 10. The method of claim 9, wherein the first polypeptide comprises or consists of SEQ ID NO: B, the second polypeptide comprises or consists of SEQ ID NO: C, and the third polypeptide comprises or consists of SEQ ID NO: A, Fc antigen domain constructs. The Fc antigen according to any one of claims 9 to 12, wherein the CH3 domain of each said Fc domain monomer comprises up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. domain constructs. 14. The method according to any one of claims 1 to 13, wherein the CH3 domain of each said Fc domain monomer is at most 8, 7, 6, 5, 4, 3, 2 or 1 compared to the amino acid sequence of a human IgG1 CH3 domain. An Fc antigen domain construct comprising a single amino acid substitution. 15. The method according to any one of claims 1 to 14, wherein each said Fc domain monomer independently has at most 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution SEQ ID NO: An Fc antigen domain construct comprising the amino acid sequence of any of 42, 43, 45, and 47. 16. The Fc antigen domain monomer according to any one of claims 1 to 15, wherein the single amino acid substitution is only in the CH3 domain. 17. The method of any one of claims 1 to 16, wherein the first Fc domain monomer and the third Fc domain monomer promote homodimerization between the first Fc domain monomer and the third Fc domain monomer. , 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution. 18. The method of any one of claims 1-17, wherein the second Fc domain monomer and the fifth Fc domain monomer promote heterodimerization between the second Fc domain monomer and the fifth Fc domain monomer at most 8. , 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution, wherein said fourth Fc domain monomer and said sixth Fc domain monomer are heterologous between said fourth and sixth Fc domain monomers. An Fc antigen domain construct comprising up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitution that promotes dimerization. 19. The Fc antigen domain construct of any one of claims 1-18, wherein the substitution promoting homodimerization is selected from the substitutions of Tables 4A and 4B. 20. The Fc antigen domain construct according to any one of claims 1 to 19, wherein the substitution promoting heterodimerization is selected from the substitutions in Table 3. A method of treating cancer or an autoimmune disease, comprising:
21. A method comprising administering the composition or construct of any one of claims 1-20. The method of claim 21 , wherein the cancer is selected from the group of indications consisting of hematologic malignancies and/or solid tumors. 22. The method of claim 21, wherein the cancer is, for example, gastric cancer, breast cancer, colon cancer, lung cancer, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, NK cell leukemia, NK/T-cell lymphoma, chronic lymphocytic leukemia, plasma cell leukemia, and multiple myeloma. 24. The method of claim 22 or 23, wherein the cancer is resistant to daratumumab or any other therapeutic anti-CD38 monoclonal antibody therapeutic. 22. The method of claim 21, wherein the autoimmune disease is an autoantibody-mediated disease, i.e. myasthenia gravis (MG), MuSK-MG, myocarditis, Lambert-Eaton, myasthenia gravis, neuromyotonia, optic neuromyelitis. , narcolepsy, acute motor axonal neuropathy, Guillain-Barré syndrome, Fisher syndrome, acute ataxia neuropathy, paraneoplastic ankylosing syndrome, chronic neuropathy, peripheral neuropathy, acute disseminated encephalomyelitis, multiple sclerosis, Goodpasture Syndrome, membranous neuropathy, glomerulonephritis, alveolar proteinosis, CIPD, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pemphigus vulgaris, deciduous pemphigus, pemphigus vesicular, pemphigus pregnancy, acquired epidermolysis bullosa, Neonatal lupus erythematosus, dermatitis herpetiformis, Graves' disease, Addson's disease, ovarian insufficiency, autoimmune orchitis, Sjogren's disease, autoimmune gastritis, rheumatoid arthritis, SLE, dry eye disease, vasculitis (acute), carditis, antibody-mediated A method selected from the group consisting of rejection. A method for treating a disorder selected from AL amyloidosis, Castleman's disease, monoclonal gamma unidentified (MGUS), biclonal gamma malignancy, osteosclerotic myeloma (POEMS syndrome), heavy chain disease, solitary plasmacytoma, extramedullary plasmacytoma, ,
21. A method comprising administering the construct or composition of any one of claims 1-20.

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