TW202216762A - TGFBR2-ECD mutant, and fusion protein comprising same and use thereof - Google Patents

Abstract

Provided are a TGFBR2-ECD mutant, a fusion polypeptide, targeting molecule or fusion protein comprising same, and the use of the mutant, fusion polypeptide, targeting molecule or fusion protein in the preparation of a drug for treating and/or preventing neoplastic diseases.

Description

TGFBR2-ECD mutant and fusion protein containing the same and application

The present invention relates to the field of biomedicine. Specifically, the present invention provides a polypeptide, which is a TGF-β RII extracellular domain (TGFBR2-ECD) mutant, a fusion protein comprising the TGFBR2-ECD mutant, and its use in the preparation of medicines.

Transforming growth factor-beta (TGF-beta) has three highly homologous isoforms, TGF-beta1, TGF-beta2, and TGF-beta3, which together belong to the TGF-beta superfamily. The high-affinity binding of TGF-β to receptors on the membrane can initiate the downstream SMAD protein phosphorylation pathway to participate in the occurrence and development of diseases.

In the process of tumor development, TGF-β regulates the abnormal expression of genes related to tumor cell proliferation, differentiation and migration, and participates in the enhancement of tumor tissue angiogenesis, cell metastasis and mesenchymal transformation and other cancer processes. ultimately exert a cancer-promoting effect. The abnormal expression of TGF-β changes the tumor tissue microenvironment, and its high expression level in vivo is associated with poor prognosis of various tumors. In colorectal cancer, SMAD activation of downstream pathways of TGF-β can initiate the expression of biomarkers associated with poor prognosis, and high expression of TGF-β1 and TGF-β3 in patients is significantly associated with poor prognosis. Studies have shown that TGF-β can promote the generation of Treg cells, inhibit the initiation and differentiation of CD8+ T cells and NK cells, and also participate in the proliferation of CAR-T that inhibits first-generation CAR-T and CD137 costimulatory signals. In addition, TGF-β is closely related to a variety of inflammatory and autoimmune diseases: in models of inflammatory bowel disease, TGF-β leads to the exacerbation of Th17 cell-related autoimmune diseases by inducing massive differentiation of Th17 cells; in asthma models , IL-13 participates in the process of pulmonary fibrosis in patients by inducing the production of TGF-β.

TGF-beta signaling is mediated by the TGF-beta receptor complex. The TGF-β receptor complex is composed of a TGF-β receptor type I (TGF-βRI) homodimer and a TGF-β type II receptor (TGF-βRⅡ) homodimer. The study found that two extracellular domains of TGF-βRⅡ (TGFBR2-ECD) directly combined with TGF-β dimer to form a complex. Therefore, TGFBR2-ECD can be used to develop TGF-β Trap, which acts to trap TGF-β, thereby blocking the TGF-β signaling pathway.

In tumor treatment, the inhibition of TGF-β in the tumor microenvironment has been recognized as an effective therapeutic target, but the efficacy of single TGF-β inhibitor in clinical studies is not good. Some bifunctional molecules comprising TGF-β Trap have been developed in the prior art, such as bifunctional molecules comprising anti-PD-L1 antibody and TGF-β Trap (see e.g. WO2018/205985 and WO2015/118175), which can block TGF simultaneously -β signaling pathway and inhibit immune checkpoints for cancer therapy. Among them, the scheme of WO2015/118175 uses wild-type TGFBR2-ECD as TGF-β Trap, and the bifunctional molecule formed by fusion with PD-L1 antibody is prone to serious degradation and breakage during the production process, which greatly affects the development of druggability. , which also poses challenges to the efficacy and safety of drugs. The scheme of WO2018/205985 obtains a more stable bifunctional molecule by truncating the N-terminal breakable region of wild-type TGFBR2-ECD, but the truncated TGFBR2-ECD may be in vivo due to the lack of the original N-terminal Sequence and form a structure that is quite different from the wild-type protein, and there are some unknown risks.

This application claims the priority of Chinese Patent Application No. 202011011927.7 filed on September 23, 2020, entitled "TGFBR2-ECD mutants and fusion proteins comprising the same, and applications", the content of which is incorporated herein by reference in its entirety.

The present invention provides a mutant of the extracellular region of TGF-βRII, which is capable of binding TGF-β and has one or more amino acid mutations at the N-terminus, so that the mutant is more efficient than the wild-type TGF-βRII extracellular region. The body contains amino acid mutations and/or improvements in hydrophilicity in the scissile region.

In one embodiment, the mutant of the present invention comprises the amino acid sequence of SEQ ID NO: 1 and carries one or more amino acid mutations in amino acids 1-24 of its N-terminus.

In some preferred embodiments, the mutants comprise amino acid mutations in each of the three regions, and at least one S or T in each of the three regions, wherein the three regions It includes: the first region QKS (amino acids 6-8), the second region NNDMI (amino acids 10-14) and the third region DNNG (amino acids 17-20).

In some embodiments, the amino acid mutation comprises amino acid substitution in each of the three regions with a hydrophilic amino acid, an N-X-S/T motif, or a combination thereof, wherein X is other than proline (P) any amino acid. Preferably, there is at least one substitution in each of the three regions. More preferably, at least two substitutions are made in each of the three regions. In some embodiments, the hydrophilic amino acid is selected from G, T, S, N, E, Y, or a combination thereof. In some embodiments, the N-X-S/T motif is N-A-S.

In some embodiments, the amino acid mutation comprises mutating the first region QKS to GGS, TGS or NAS, the second region NNDMI to GGSGI, TGSGI or NASGI, and the third region DNNG to GGSG, TGSG or GNAS . In a specific embodiment, the mutant of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6.

In other preferred embodiments, the mutants comprise amino acid mutations in each of the three regions, and at least one S or T in each of the three regions, and further comprise Amino acid mutations in the upstream and downstream regions of the three regions. In these embodiments, the amino acid mutation comprises substituting at least one amino acid in each of the three regions with G, S, E, T, or a combination thereof, and substituting T, Y, or a combination thereof in each region Substitute at least one amino acid in the upstream and downstream regions. In some embodiments, the amino acid mutation comprises mutating the first amino acid I to T, amino acids 5-8 (VQKS) to TQES or TQTS, and amino acids 9-15 (VNNDMIV) to TSESMIT or TNNSMIT, and mutating amino acids 17-24 (DNNGAVKF) to GESGATKY or GTSGATKY. In a specific embodiment, the mutant has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

In another aspect, the present invention provides a targeting molecule comprising a mutant of the present invention and a targeting moiety, wherein the targeting moiety is covalently linked to the N-terminus of the mutant, and the targeting moiety targets The targeted molecules are selected from: growth factors and their receptors, cytokines and their receptors, cell surface antigens, immune checkpoint molecules, hormones and other biologically active components in vivo. In one embodiment, the targeting moiety is an antibody or antigen-binding fragment thereof, mimetibody, ligand, ligand binding domain or functionally active domain of an in vivo active protein. In one embodiment, the targeting moiety is an Fc fragment, preferably the Fc fragment of human IgGl. In one embodiment, the Fc fragment of human IgGl is fused to the N-terminus of the TGFBR2-ECD mutants of the invention to provide an Fc-TGFBR2-ECD fusion protein. In a specific embodiment, the Fc-TGFBR2-ECD fusion protein comprises the amino acid sequence of SEQ ID NO:52.

In a specific embodiment, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof.

In another aspect, the present invention provides a fusion polypeptide comprising the mutant of the present invention and a first polypeptide, and the C-terminus of the first polypeptide is covalently linked to the N-terminus of the mutant. In one embodiment, the first polypeptide comprises at least one heavy chain variable region of an antibody or at least one light chain variable region of an antibody. In a specific embodiment, the antibody is an anti-PD-L1 antibody.

In yet another aspect, the present invention provides a fusion protein comprising a fusion polypeptide of the present invention and a second polypeptide, wherein the second polypeptide, when combined with the first polypeptide, forms a targeting moiety. In one embodiment, the first polypeptide comprises at least one heavy chain variable region of an antibody and the second polypeptide comprises at least one light chain variable region of an antibody. In yet another embodiment, the first polypeptide comprises at least one light chain variable region of an antibody and the second polypeptide comprises at least one heavy chain variable region of an antibody. In a specific embodiment, the antibody is an anti-PD-L1 antibody. In a specific embodiment, the fusion proteins of the invention form dimers.

In another aspect, the present invention also provides a pharmaceutical composition comprising the targeting molecule, fusion polypeptide or fusion protein of the present invention, and a pharmaceutically acceptable carrier. The present invention also provides the use of the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition in the preparation of a medicament for the treatment and/or prevention of neoplastic diseases.

In yet another aspect, the present invention also provides nucleic acid molecules encoding the mutants, targeting molecules, fusion polypeptides or fusion proteins, expression vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules or expression vectors.

definition

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are the terms and routine procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, "at least one (species)" or "one (species) or more (species)" can mean 1, 2, 3, 4, 5, 6, 7, 8 (species) or more ( kind).

As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetically (eg, recombinantly) produced, that specifically bind to an epitope of an antigen through at least one antigen-binding site. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to the antigen binding domain of an immunoglobulin (the antibody binding site). Antibodies encompass antigen-binding fragments. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single domain antibodies Antibodies and antigen-binding fragments such as, but not limited to, Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single chain Fv (scFv) ), single-chain Fab (scFab), diabody, disulfide-linked Fab (dscFab), or an antigen-binding fragment of any of the foregoing. Antibodies provided herein include any immunoglobulin class (eg, IgG, IgM, IgD, IgE, IgA, and IgY), any class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (eg, IgG2a) and IgG2b).

As used herein, an "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but that comprises at least a portion of the variable region (eg, one or more CDRs and/or one or more of the variable region of said antibody that binds an antigen) antigen-binding sites), and thus retain at least a portion of the ability of the full-length antibody to specifically bind antigen. Antigen-binding fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as derivatives produced by chemical synthesis or genetic engineering techniques (eg, recombinant DNA techniques). Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see , eg Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers. Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding regions), results in an antibody that immunospecifically binds an antigen.

"Traditional" or "full-length" antibodies typically contain four polypeptides: two heavy chains and two light chains. Each chain may contain variable and constant regions. Each light chain comprises a light chain variable region ( _VL ) and a light chain constant region ( _CL ) from the N-terminus to the C-terminus. Each heavy chain includes a heavy chain variable region (V _H ) and one or more heavy chain constant regions ( _CH ) from the N-terminus to the C-terminus, and the heavy chain constant region from the N-terminus to the _C -terminus may include CH1, _CH2 and _CH3 , or _CH1 , _{CH2, CH3 and CH4} . Each _VL and each _VH may contain three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)". Herein, the CDRs of the light chain variable region may be referred to as LCDR1, LCDR2 and LCDR3, and the CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2 and HCDR3. One of skill in the art can identify CDRs using methods well known in the art, such as using Kabat or Chothia coding (see e.g. Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services , NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol . 196:901-917). An Fv fragment comprising six CDRs (i.e. three heavy chain CDRs and three light chain CDRs) is generally considered to be the smallest unit of an antigen binding site, but in some cases less than six CDRs (e.g. three, Four or five) other fragments (such as single domain antibodies, also known as nanobodies) also have the ability to bind antigen.

As used herein, an "Fv fragment" refers to an antigen-binding fragment comprising a pair of VH and VL. "Single-chain Fv (scFv)" can be obtained by linking VH and VL via a peptide linker. "Disulfide stabilized Fv (dsFv)" or "single-chain disulfide stabilized Fv (scdsFv or dsscFv)" can be obtained by introducing a disulfide bond into Fv or scFv, respectively.

As used herein, "Fab" comprises one complete antibody light chain (VL-CL) and antibody heavy chain variable region and one heavy chain constant region (VH-CH1, also known as Fd). A single-chain "Fab (scFab)" can be obtained by linking CL and CH1 in "Fab" with a peptide linker. "F(ab') ₂ " essentially comprises two Fab fragments linked by disulfide bonds in the hinge region. "Fab'" is one half of F(ab') ₂ , which can be obtained by reducing disulfide bonds in the hinge region of F(ab') ₂ .

As used herein, an "Fc fragment" refers to a papain-digested crystallizable fragment of a full-length antibody. In general, the Fc fragment of an IgG may comprise part of the hinge region, CH2 and CH3. As used herein, an Fc fragment may comprise at least part of the hinge region (eg all or part of the hinge region), CH2 and CH3. Those skilled in the art can judge the positions of CDR, FR, VH, VL, CL, CH1, CH2, CH3 and hinge regions in the antibody according to known algorithms and software. For the description of applicable algorithms and software, see for example William R. Strohl, Lila M. Strohl, (2012), Therapeutic Antibody Engineering, Woodhead Publishing, pp. 37-56.

As used herein, a "diabody" refers to an antibody comprising two scFvs with a short peptide linker (approximately 5-10 amino acid residues) between the _VH and _VL in each scFv Linked such that the _VH and _VL interchain pairings (ie, the _VH of the first scFv and the _VL pairing of the second scFv, the _VL pairing of the first scFv and the _VH pairing of the second scFv) form the antigen binding site.

As used herein, a "chimeric antibody" refers to an antibody in which a portion (eg, CDRs, FRs, variable regions, constant regions, or a combination thereof) is identical or homologous to corresponding sequences in an antibody derived from a particular species, and the remainder The portion is identical or homologous to the corresponding sequence in an antibody derived from another species. Chimeric antibodies can be produced by antibody engineering. Methods of antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be generated by recombinant DNA techniques (eg see Sambrook, J., et al. (1989). Molecular cloning: a laboratory manual , 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody has been modified to increase sequence homology to a human antibody. Humanized antibodies can be obtained by antibody engineering any antibody of a non-human species or an antibody (eg, a chimeric antibody) that contains sequences derived from a non-human species therein. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art.

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of human antibody encompasses whole or full-length antibodies, fragments thereof and/or antibodies comprising at least one human heavy and/or light chain polypeptide.

As used herein, "single domain antibodies (sdAbs)" also referred to as "nanobodies" refer to antibodies comprising a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable regions of full-length antibodies, single variable domains typically comprise CDR1, CDR2, and CDR3 that form the antigen-binding site, as well as supporting framework regions.

"Affinity" or "binding affinity" is used to measure the strength of mutual binding between a molecule and its ligand through non-covalent forces, such as the binding strength between a targeting moiety of the invention and its target, such as an antibody or antigen-binding fragment Binding strength to antigen or between TGFBR2-ECD mutants of the invention and TGF-beta. The magnitude of affinity is usually reported as the equilibrium dissociation constant _KD , often determined by measuring the association constant (ka) and the dissociation constant (kd) and calculating the quotient of kd divided by ka ( _KD = kd/ka). KD can be readily determined using conventional techniques, such as by equilibrium dialysis; by using the Octet _RED96 detection system, using the general methods listed by the manufacturer; by using an enzyme-linked immunosorbent assay (ELISA); by using radiolabeled target antigens radioimmunoassay; or by other methods known to the skilled artisan.

"Specific binding" means that two molecules bind to each other with higher affinity. Generally, the _KD value between the two molecules that specifically bind can be 10 ^-6 , 10 ^-7 , 10 ^-8 to 10 ^-9 M or lower. For example, an antibody or antigen-binding fragment can specifically bind with a _KD value of ^10-6 to ^10-9 M between the antigen-binding site and the antibody-binding site of the antigen, and the relationship between TGFBR2-ECD and TGF-β can be Specific binding with _KD values of ^10-6 to ^10-9 M.

In polypeptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al. , Molecular Biology of the Gene , 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).

As used herein, the term "polypeptide" refers to a polymer comprising at least two amino acids or derivatives thereof linked by peptide bonds. A "protein" may be formed covalently or non-covalently from one or more polypeptides.

As used herein, the term "mutation" refers to a polypeptide or protein comprising a substitution, deletion or addition of one or more amino acids. The substitutions can be conservative or non-conservative. In embodiments of the invention, one amino acid may be substituted for another amino acid (eg, G for Q), or one amino acid motif may be substituted for another amino acid motif (eg, N-A-S motif for Q-K-S motif). Herein, the event of substituting an amino acid motif at a position or an amino acid at a position is described as a substitution. Herein, when specifying a mutation position, the n-th amino acid refers to the n-th amino acid counted from the first amino acid at the amino terminus (ie, the N-terminus) of the polypeptide. Herein, the assignment of mutation positions is based on SEQ ID NO: 1.

As used herein, a "wild-type" polypeptide or protein refers to a naturally occurring polypeptide or protein that has not been engineered. A "mutant" or "mutant" of a polypeptide or protein carries a mutation relative to the "wild-type" polypeptide or protein. For example, the extracellular region of wild-type TGF-betaRII comprises the sequence of SEQ ID NO:1. Mutants in the extracellular domain of TGF-betaRII carry mutations relative to the sequence of SEQ ID NO:1.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

A polypeptide, protein or nucleic acid molecule of the invention may be "isolated". The expression "isolated" means that the polypeptide, protein or nucleic acid molecule may, for example, be engineered and/or separated from other substances in its naturally occurring environment. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.

As used herein, "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. The level of expression of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining or silver staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express nucleic acids or polypeptides encoded by vectors. Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a vehicle used to introduce exogenous nucleic acid into a host cell, which is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acid encoding a polypeptide. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like. As used herein, a vector can be expressed or replicated in a host cell.

As used herein, an "expression vector" includes a vector capable of expressing a polypeptide comprising a polynucleotide sequence encoding a polypeptide of interest. The polynucleotide sequence encoding the polypeptide of interest is operably linked to regulatory sequences capable of affecting its expression. Such regulatory sequences may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector may refer to a recombinant DNA or RNA construct, such as a plasmid, phage vector, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA or RNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.

As used herein, the term "targeting moiety" refers to a molecule capable of specifically binding a target. A targeting moiety can target a molecule to which it is covalently attached (eg, a TGFBR2-ECD mutant of the invention) to a specific target. A targeting moiety can recognize one or more targets.

As used herein, the term "ligand" refers to an active substance capable of regulating a specific cellular physiological activity, which can bind to a specific in vivo active protein to initiate a physiological and biochemical reaction in the cell.

As used herein, "in vivo active protein" refers to the ligand itself as well as a protein that can modulate cellular physiological activities, such as a protein that accepts an extracellular or intracellular ligand and mediates signal transduction, which can have a ligand binding domain and/or function Sexual domain. In vivo active proteins can be ligands, receptors, kinases, ion channels or other proteins involved in signal transduction. As used herein, the term "ligand binding domain" refers to a domain in an active protein in vivo that is capable of specifically recognizing and binding a ligand. "Functional domain" refers to a domain in an active protein in vivo that can mediate signal transduction and initiate physiological and biochemical reactions in cells.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to an article. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder. Also, as used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when applied to an object, will have the desired prophylactic effect, eg, preventing or delaying disease or the occurrence or recurrence of symptoms, reducing the likelihood of the occurrence or recurrence of disease or symptoms. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

As used herein, the term "individual" refers to a mammal, such as a human.

TGFBR2-ECD mutant

In one aspect, the present invention provides a mutant of the extracellular domain of TGF-βRII (TGFBR2-ECD), which is capable of binding TGF-β and has one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid mutations such that the mutants comprise amino acids in the breakable region compared to the extracellular region of wild-type TGF-βRII Mutations and/or improvements in hydrophilicity.

As used herein, the terms "TGF-betaRII extracellular domain" or "TGFBR2-ECD" have the same meaning and refer to the extracellular domain of the TGF-beta type II receptor (TGF-betaRII), which has the ability to bind TGF-beta active. Dimerized TGFBR2-ECD can bind and trap TGF-β dimers as a TGF-β Trap (see, eg, WO2015/118175 and WO2018/205985), thereby inhibiting TGF-β signaling and abrogating TGF-β signaling-associated immunity inhibition. Inhibition of the TGF-beta signaling pathway can be determined using various assays known in the art, eg, detecting the expression of downstream genes controlled by the TGF-beta signaling pathway.

Wild-type TGFBR2-ECD is a 136 amino acid peptide (SEQ ID NO: 1) that can be combined with other polypeptides to form bifunctional molecules comprising TGF-β Trap, such as anti-PD-L1/TGF-β Trap (see e.g. WO2015/118175 and WO2018/205985). However, bifunctional molecules containing wild-type TGFBR2-ECD are prone to breakage, which is not conducive to subsequent druggability development. The TGFBR2-ECD mutants of the present invention are improved polypeptides relative to wild-type TGFBR2-ECD. Compared with the bifunctional molecule comprising the wild-type TGFBR2-ECD, the bifunctional molecule comprising the TGFBR2-ECD mutant of the present invention is less likely to be degraded during the production process and has better druggability.

The inventors found that wild-type TGFBR2-ECD is prone to cleavage in the N-terminal amino acids 1-24. In one embodiment, the mutant comprises the amino acid sequence of SEQ ID NO: 1 with one or more amino acid mutations in its N-terminal amino acids 1-24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 9 or more. In another embodiment, the number of amino acid mutations is 16 or less.

In some preferred embodiments, the mutants comprise amino acid mutations in at least one, preferably each, of the three regions. Preferably the mutant contains at least one S or T in each of the three regions. The three regions include: the first region QKS (amino acids 6-8), the second region NNDMI (amino acids 10-14), and the third region DNNG (amino acids 17-20).

In some embodiments, the amino acid mutation comprises amino acid substitution in each of the three regions with a hydrophilic amino acid, an N-X-S/T motif, or a combination thereof, wherein X is other than proline (P) any amino acid. Preferably, there is at least one substitution in each of the three regions. More preferably, at least two substitutions are made in each of the three regions. In some embodiments, the hydrophilic amino acid is selected from G, T, S, N, E, Y, or a combination thereof. In some embodiments, the N-X-S/T motif is N-A-S.

In some embodiments, the amino acid mutation comprises mutating the first region QKS to GGS, TGS or NAS, the second region NNDMI to GGSGI, TGSGI or NASGI, and the third region DNNG to GGSG, TGSG or GNAS . In a specific embodiment, the mutant of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6.

In other preferred embodiments, the mutants comprise amino acid mutations in each of the three regions, and at least one S or T in each of the three regions, and further comprise Amino acid mutations in the upstream and downstream regions of the three regions. In these embodiments, the amino acid mutation comprises substituting at least one amino acid in each of the three regions with G, S, E, T, or a combination thereof, and substituting T, Y, or a combination thereof in each region Substitute at least one amino acid in the upstream and downstream regions. In some preferred embodiments, the amino acid mutation comprises mutating the first amino acid I to T, 5-8 amino acids (VQKS) to TQES or TQTS, 9-15 amino acids (VNNDMIV) is TSESMIT or TNNSMIT, and amino acids 17-24 (DNNGAVKF) are mutated to GESGATKY or GTSGATKY. In a specific embodiment, the mutant has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

targeting molecule

In yet another aspect, the present invention provides a targeting molecule comprising a TGFBR2-ECD mutant of the present invention and a targeting moiety covalently linked to the N-terminus of the mutant. In one embodiment, the targeting moiety targets a target selected from the group consisting of growth factors and their receptors, cytokines and their receptors, cell surface antigens, immune checkpoint molecules, hormones, and other biologically active components in vivo . The targeting molecule of the present invention is also called a bifunctional molecule, which can play a dual role of simultaneously blocking the TGF-β signaling pathway and specifically binding to a specific target.

The targeting moiety and the TGFBR2-ECD mutant can be covalently linked in any manner to form the targeting molecule. For example, one or more polypeptides in the targeting moiety can be covalently linked to the mutant in the form of recombinant polypeptides or proteins using DNA recombination technology, or the targeting moiety can be covalently linked by chemical coupling or enzymatic coupling methods. price connection.

In one embodiment, the targeting moiety comprises one or more polypeptides, and wherein the C-terminus of at least one polypeptide is covalently linked to the N-terminus of the mutant to form a fusion polypeptide.

In some embodiments, the targeting molecule further comprises a linker between the targeting moiety and the mutant. Preferred linkers are peptide linkers. Preferably, the linker is (G ₄ S) _n G, wherein n is an integer of 2-6, preferably an integer of 3-5, more preferably 4.

targeting moiety

A targeting moiety can specifically bind to its target. In preferred embodiments, targeting moieties may target tumor specific markers, tumor stem cells, tumor microenvironment, immune checkpoints, or tumorigenesis regulatory mechanisms. Targets of targeting moieties may include, for example, growth factors or their receptors, cytokines or their receptors, cell surface antigens, immune checkpoint molecules, hormones, or other biologically active components in vivo, and the like. Immune checkpoint molecules can include, for example, PD1, CTLA4, LAG-3, BTLA, TIM-2, LAIR1, PD-L1, B7-DC, B7-H3, B7-H4, HVEM, and TIM-4. In a preferred embodiment, the targeting moiety targets the tumor microenvironment, eg, a cytokine or its receptor or a growth factor or its receptor as described herein can be targeted. Examples of targets of targeting moieties may include, but are not limited to: CD13, CD19, CD22, CD25, CD27, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352, CLDN18.2, DLL3, CN33, GPNMB, ENPP3, Nectin-4, HER2/ErbB2, EGFR, FGFR, VEGFR, HGFR, PDGFR, VEGF, HGF, FGF, FGF- 2. PDGF, EGF, Galectin-3, SLC44A4/AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, SLITRK6, SLAMF7/CS1, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, CA6, Integrin αV, TDGF1, Ephrin A4, PTK7, NOTCH3, C4.4A, FLT3, PD1, CTLA4, LAG-3, BTLA, TIM-2, LAIR1, PD-L1, B7 - DC, B7-H3, B7-H4, HVEM and TIM-4.

Targeting moieties that can be used in the present invention include, but are not limited to, for example, antibodies or antigen-binding fragments thereof, mimetibodies, ligands, ligand binding domains or functionally active domains of in vivo active proteins, and the like. In some embodiments, the targeting moiety is a ligand. In one embodiment, the ligand is a cell surface antigen, cytokine, growth factor or hormone. Cell surface antigens can include tumor-specific antigens and tumor-associated antigens. Cytokines may be selected from: G-CSF, GM-CSF, eotaxin/CCL11, MCP-1/CCL2, MIP-1α/CCL4, RANTES/CCL5, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, INFα, INFβ, INFγ and TNFα and TNFβ. Growth factors may be selected from: VEGF, HGF, FGF, FGF-2, PDGF, IGF, TGF, NGF, EPO and EGF. The hormone may be a peptide hormone or a steroid hormone, preferably a peptide hormone. In some embodiments, the targeting moiety is the ligand binding domain of an in vivo active protein. In vivo active proteins can be receptors (eg, T cell receptors or their co-receptors, cytokine receptors, growth factor receptors, hormone receptors, etc.), kinases, ion channels, or other proteins involved in signal transduction. Preferably, the ligand binding domain or functional domain is a ligand binding domain or functional domain of a T cell receptor, cytokine receptor, growth factor receptor, hormone receptor. The ligand, ligand binding domain or functionally active domain can be wild-type or mutant.

In one embodiment, the targeting moiety is an antibody or antigen-binding fragment thereof, mimetibody, ligand, ligand binding domain or functionally active domain of an in vivo active protein.

In some embodiments, the targeting moiety is an Fc fragment. Preferably, the targeting moiety is the Fc fragment of human IgGl. Fc fragments can be modified to achieve the desired in vivo activity, such as prolonging the serum half-life of the targeted molecule, or to enhance or attenuate the effector functions of the targeted molecule (such as antibody-dependent cell-mediated toxicity (ADCC) and complement-dependent cytotoxicity) effect (CDC)). In some embodiments, the Fc fragment of human IgGl comprises the N297A mutation, ie the asparagine (N) at position 297 of the human IgGl heavy chain is mutated to alanine (A). The Fc fragment containing the N297A mutation lost its ability to bind to FcγR, thereby abrogating Fc fragment-mediated ADCC. In one embodiment, the Fc fragment of human IgGl comprises the amino acid sequence of SEQ ID NO:53. In one embodiment, the Fc fragment of human IgGl is fused to the N-terminus of the TGFBR2-ECD mutants of the invention, optionally via a linker, to provide an Fc-TGFBR2-ECD fusion protein. In a specific embodiment, the Fc-TGFBR2-ECD fusion protein comprises the amino acid sequence of SEQ ID NO:52.

Preferably, the targeting moiety is an antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof that specifically binds to the above-mentioned target. The antibody or antigen-binding fragment thereof may be selected from the group consisting of synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single domain antibodies , Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, dsFv fragment, Fd fragment, Fd' fragment, scFv, scFab, diabody. In a preferred embodiment, the targeting moiety is an antibody or antigen-binding fragment thereof that targets the tumor microenvironment. In a specific embodiment, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof. The anti-PD-L1 antibodies include, but are not limited to, MSB0010718C, MEDI4736, BMS-936559, MPDL3280A, and the anti-PD-L1 antibodies described in WO 2019/233462.

In some embodiments, the targeting moiety comprises an anti-PD-L1 antibody or antigen-binding fragment thereof. Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof is as described above.

In one embodiment, the targeting moiety comprises the _VH and _VL of the antibody, and the C-termini of _VH and _VL are each covalently linked to the N-terminus of the two mutants. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein.

In one embodiment, the targeting moiety comprises the _VH and _VL of the first antibody and the _VH and _VL of the second antibody, and the C-termini of the two _VHs are each covalently linked to the N-terminus of the two mutants . The first antibody and the second antibody can be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

In one embodiment, the targeting moiety comprises the _VH - _CH1 and light chains of the antibody, and the _C -termini of CH1 and _CL are each covalently linked to the N-termini of the two mutants. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein.

In one embodiment, the targeting moiety comprises the _VH -CH1 and light chain of the first antibody and the _VH - _CH1 and light chain of the second antibody, and the _C -terminus of both _VH - _CH1 Each is covalently linked to the N-terminus of the two mutants. The first antibody and the second antibody can be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

In one embodiment, the targeting moiety comprises the heavy and light chains of the first antibody and the heavy and light chains of the second antibody, and the C-termini of the two heavy chains are each covalently linked to the N-termini of the two mutants . The first antibody and the second antibody can be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

Anti-PD-L1 antibody

In some preferred embodiments, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof. Exemplary anti-PD-L1 antibodies include, but are not limited to, MSB0010718C, MEDI4736, BMS-936559, MPDL3280A, and the anti-PD-L1 antibodies described in WO 2019/233462. In some preferred embodiments, the anti-PD-L1 antibodies are the anti-PD-L1 antibodies B10, H12 and B11 described in WO 2019/233462, the relevant contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-L1 antibody is a B10 antibody comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; The light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence of SEQ ID NO:22.

In yet another embodiment, the anti-PD-L1 antibody is a B11 antibody comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 26; The light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 28, LCDR2, which comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence of SEQ ID NO:30.

In another embodiment, the anti-PD-L1 antibody is an H12 antibody comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 34; The light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 36, LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence of SEQ ID NO:38.

In yet another embodiment, the B10 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19. In yet another embodiment, the B10 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:23. In one embodiment, the B11 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27. In yet another embodiment, the B11 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:31. In one embodiment, the H12 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:35. In yet another embodiment, the H12 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:39.

In yet another embodiment, the B10, B11 or H12 antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:40. In yet another embodiment, the B10, B11 or H12 antibody further comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:41.

In one embodiment, the B11 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the B11 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43.

fusion polypeptide

In another aspect, the present invention provides a fusion polypeptide comprising a TGFBR2-ECD mutant and a first polypeptide, wherein the C-terminus of the first polypeptide is covalently linked to the N-terminus of the mutant.

In some embodiments, the first polypeptide is a targeting moiety as described above. In some embodiments, the first polypeptide forms a targeting moiety as described above with other polypeptides.

In some embodiments, the first polypeptide comprises at least one heavy chain variable region of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises a heavy chain variable region of an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 27 and 35. In yet another specific embodiment, the first polypeptide further comprises a heavy chain constant region of an antibody, the heavy chain constant region comprising the amino acid sequence of SEQ ID NO:41.

In some embodiments, the first polypeptide comprises the heavy chain of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises the heavy chain of an antibody, the heavy chain comprising the amino acid sequence of SEQ ID NO:43.

In some embodiments, the first polypeptide comprises at least one light chain variable region of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises a light chain variable region of an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 31 and 39. In yet another specific embodiment, the first polypeptide further comprises a light chain constant region of an antibody, the light chain constant region comprising the amino acid sequence of SEQ ID NO:40.

In some embodiments, the first polypeptide comprises the light chain of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises the light chain of an antibody, the light chain comprising the amino acid sequence of SEQ ID NO:42.

In some embodiments, the fusion polypeptide further comprises a linker between the first polypeptide and the mutant. Preferred linkers are peptide linkers. Preferably, the linker is (G ₄ S) _n G, wherein n is an integer of 2-6, preferably an integer of 3-5, more preferably 4.

In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-15.

fusion protein

In yet another aspect, there is provided a fusion protein comprising the fusion polypeptide of the present invention and a second polypeptide. The fusion protein of the present invention may be a targeting molecule as described above, comprising a targeting moiety and a mutant of the present invention. In one embodiment, the second polypeptide, when combined with the first polypeptide, forms a targeting moiety as described herein.

In some embodiments, the first polypeptide comprises at least one heavy chain variable region of an antibody and the second polypeptide comprises at least one light chain variable region of an antibody. In some embodiments, the first polypeptide comprises at least one light chain variable region of an antibody and the second polypeptide comprises at least one heavy chain variable region of an antibody.

In a specific embodiment, the first polypeptide comprises an antibody heavy chain and the second polypeptide comprises an antibody light chain.

In one embodiment, the antibody is an anti-PD-L1 antibody or antigen-binding fragment thereof described herein. Therefore, the present invention also provides a bifunctional fusion protein targeting PD-L1 and TGF-β, which can also be called mutant anti-PD-L1/TGF-β Trap.

In one embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence of SEQ ID NO:22.

In yet another embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, HCDR3 comprising the amino acid sequence of SEQ ID NO: 26; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 28, LCDR2, which comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence of SEQ ID NO:30.

In another embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 34; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 36, LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence of SEQ ID NO:38.

In one embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

In yet another embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:31.

In another embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:39.

In yet another embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region and a heavy chain constant region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 27 and 35, the heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain variable region and a light chain constant region, the light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 31 and 39, the light chain constant region Contains the amino acid sequence of SEQ ID NO:40.

In a specific embodiment, the fusion protein comprises: (1) A fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain comprising the amino acid sequence of SEQ ID NO:42.

In some embodiments, the fusion protein further comprises a linker between the first polypeptide and the mutant, preferably, the linker is (G ₄ S) _n G, wherein n is 2-6 Integer, preferably an integer from 3 to 5, more preferably 4.

In one embodiment, the fusion protein forms a dimer.

neoplastic disease

The targeting molecules, fusion polypeptides, fusion proteins and pharmaceutical compositions of the present invention can be used to treat and/or prevent tumors, inflammation, autoimmunity and other diseases caused by TGF-β family related pathways.

The present invention also relates to the use of the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition of the present invention in the preparation of a medicament for the treatment and/or prevention of neoplastic diseases.

The present invention also relates to a method for treating and/or preventing neoplastic diseases, which comprises administering the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition of the present application to an individual in need.

The targeting molecule, fusion polypeptide, fusion protein (especially anti-PD-L1/TGF-β Trap) or pharmaceutical composition of the present invention can be used for the treatment and/or prevention of neoplastic diseases. Preferred neoplastic diseases (or cancers) that can be prevented and/or treated include cancers generally responsive to immunotherapy, particularly PD-L1 positive cancers. Non-limiting examples of treatable cancers include, but are not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioblastoma tumor, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumor and osteosarcoma. Examples of other cancers include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva Cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus, small bowel, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, urethra, penis, chronic or acute leukemia , including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system Systemic (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmental Induced cancers, including asbestos-induced cancers, and combinations of such cancers. The fusion protein and pharmaceutical composition of the present invention can also be used to treat metastatic cancer, especially metastatic cancer expressing PD-L1. In yet another embodiment, the neoplastic disease (or cancer) is lung cancer, ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, Glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer or osteosarcoma, especially colorectal cancer or non-small cell lung cancer.

In some embodiments, the anti-PD-L1/TGF-β Traps of the present invention can be used to treat cancer. In some embodiments, the cancer is a PD-L1 positive cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasal Pharyngeal cancer, laryngeal cancer, cervical cancer, endometrial cancer, osteosarcoma, colorectal cancer and non-small cell lung cancer.

Nucleic Acids, Vectors and Host Cells

In another aspect, the present invention provides isolated nucleic acid molecules encoding mutants, targeting molecules, fusion polypeptides or fusion proteins of the invention. The nucleotide sequence of the nucleic acid molecule can be codon-optimized for the host cell used for expression.

The present invention also provides an expression vector comprising at least one nucleic acid molecule of the present invention.

One aspect of the present invention also provides a host cell comprising at least one nucleic acid molecule or expression vector of the present invention.

Production method of fusion polypeptide

Yet another aspect of the present invention also relates to a method for producing the mutant, targeting molecule, fusion polypeptide or fusion protein of the present invention, the method comprising: (i) culturing the host cell of the invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and (ii) isolating and purifying the mutant, targeting molecule, fusion polypeptide or fusion protein of the present invention.

pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier. Preferably, the drug is used to treat and/or prevent tumors, inflammation, autoimmunity and other diseases caused by TGF-β family related pathways. More preferably, the medicament is for the treatment and/or prevention of neoplastic diseases as described above.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, an active compound, such as a fusion protein of the present invention, can be encapsulated in a material to protect it from the action of acids and other natural conditions that can inactivate the compound.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, EDTA (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable drug can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of these media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except to any extent incompatible with the active compound, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) from previously sterile-filtered solutions thereof to obtain a powder of the active ingredient plus any additional desired ingredient.

The amount of active ingredient that can be combined with carrier materials to prepare a single dosage form will vary depending upon the item being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Typically, on a 100% basis, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient, and is pharmaceutically acceptable carrier combination.

Dosage regimens can be adjusted to provide the best desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the items to be treated; each unit containing a predetermined quantity of active compound calculated to be in association with the required pharmaceutical carrier produce the desired therapeutic effect. The specific description of dosage unit forms of the present invention is limited to and depends directly on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent in the art for formulating such for the treatment of individual sensitivities. Restriction of Active Compounds.

For administration of the fusion proteins or pharmaceutical compositions of the invention, dosages range from about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of the subject's body weight. For example, the dose can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or in the range of 1-20 mg/kg body weight Inside. Exemplary treatment regimens require dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or with a shorter initial dosing interval. The short-term dosing interval was lengthened. The mode of administration can be intravenous drip.

Alternatively, the tumor-targeted fusion protein can also be administered as a sustained release formulation, in which case less frequent dosing is required. Dosage and frequency vary according to the half-life of the drug molecule in the patient. The dose and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively lower doses are administered at less frequent intervals over an extended period of time. Some patients continue to be treated for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer higher doses at shorter intervals until the progression of the disease is lessened or stopped, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient can be administered on a prophylactic regimen.

The actual dosage level of the active ingredient in the pharmaceutical composition may vary to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The dose level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular combination employed. other drugs, compounds and/or materials with which the drug is to be used in combination, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

An "effective amount" refers to a dose sufficient to reduce the severity of symptoms of the disease, increase the frequency and duration of asymptomatic periods of the disease, or prevent damage or disability due to the suffering of the disease. For example, for the treatment of tumors, an "effective amount" of a fusion protein or pharmaceutical composition of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, and more Preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can also be assessed by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. An effective amount of the fusion protein or pharmaceutical composition of the present invention can reduce tumor size, or otherwise alleviate symptoms of the object, such as preventing and/or treating metastasis or recurrence. One skilled in the art can determine this amount based on factors such as the size of the article, the severity of the article's symptoms, and the particular composition or route of administration chosen.

The fusion proteins or pharmaceutical compositions of the present invention can be administered by one or more routes of administration using one or more methods known in the art. It will be understood by those skilled in the art that the route and/or mode of administration will vary depending on the desired result. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the fusion proteins or pharmaceutical compositions of the present invention may also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, eg, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those skilled in the art.

The fusion proteins of the present invention may also be conjugated to therapeutic moieties such as cytotoxins, radioisotopes or biologically active proteins.

Cytotoxins include any agent that is detrimental to cells (eg, kills cells).

Examples include, but are not limited to: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecacin, mitomycin, epipodophyllin glucopyranoside, epipodophyllin thiophene glycoside, vincristine Columbine, Vinca, Colchicine, Doxorubicin, Daunorubicin, Dihydroxyanthraxdione, Mitoxantrone, Spotomycin, Actinomycin D, 1-Dehydrotestosterone, Glucocorticoids, Procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs.

Therapeutic agents that can be used for conjugation also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminoenimidamine), alkylating agents (eg, chlorambucil, chlorambucil, chlorambucil, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, Streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (eg, actinomycin D (previously known as actinomycin), bleomycin, fecomycin, and atramycin (AMC)), and antimitotics (eg, vinblastine and Changchun Kui).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the fusion proteins of the present invention include duocarmycin, calicheamicin, maytansine, auristatin, and derivatives thereof.

Cytotoxins can be conjugated to the fusion proteins of the invention using linker technology used in the art, eg, to a targeting moiety (eg, an antibody) in the fusion protein. Examples of the types of linkers that have been used for cytotoxin conjugation include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. Linkers can be selected, for example, within the lysosomal compartment that are cleaved by low pH or by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D) .

The fusion proteins of the present invention can also be conjugated with radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioconjugates. Examples of radioisotopes that can be conjugated to fusion proteins of the present invention include, but are not limited to, iodine 131, indium 111, yttrium 90, and yttrium 177. Methods for preparing radioconjugates are well known in the art.

The fusion proteins of the present invention can also be conjugated to proteins having the desired biological activity. Such biologically active proteins include, for example: toxins with enzymatic activity or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon - gamma; or biological response modifiers such as immunomodulatory factors such as lymphokines, interleukins (eg IL-1, IL-2, IL-6, IL-10), chemokines (eg ccl3, ccl26, cxcl7) , granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other immunomodulatory factors such as IFN.

combination therapy

The fusion proteins or pharmaceutical compositions of the present invention can be administered in combination with chemotherapeutic agents or antibodies targeting other tumor antigens. The fusion protein of the present invention and the chemotherapeutic agent or antibody targeting other tumor antigens may be administered all at one time or separately. When administered separately (in the case of mutually different administration regimens), they may be administered continuously without interruption or at predetermined intervals. The fusion proteins of the present invention or the pharmaceutical compositions of the present invention may also be combined with radiotherapy, eg, comprising administering ionizing radiation to the patient prior to, during, and/or after the administration of the fusion proteins or pharmaceutical compositions of the present invention process.

Reagent test kit

Also included within the scope of the invention are kits comprising the mutants, fusion polypeptides, targeting molecules, fusion proteins, or nucleic acid molecules or vectors encoding the same of the invention, or combinations thereof, and instructions for use. Kits generally include a label indicating the intended use and/or method of use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

beneficial effect

Bifunctional molecules containing wild-type TGFBR2-ECD are prone to breakage, which is not conducive to subsequent druggability development. However, the bifunctional molecule containing truncated TGFBR2-ECD may form a structure that is quite different from the wild-type protein in the in vivo environment due to the lack of the original sequence of the N-terminus, and there are some unknown risks.

Aiming at the fragile nature of wild-type TGFBR2-ECD, the inventor adopted a mutant optimization strategy that is completely different from the existing technology (such as WO2018/205985), and kept the original sequence of TGFBR2-ECD as much as possible on the basis of solving the risk of druggability .

Compared to wild-type TGFBR2-ECD, the TGFBR2-ECD mutants of the present invention contain amino acid mutations in the breakable regions (QKS, NNDMI and DNNG) and/or improvements in hydrophilicity, but do not affect the affinity activity for TGF-β1 . Furthermore, the TGFBR2-ECD mutants of the present invention (particularly SEQ ID NOs: 2-8), compared to wild-type TGFBR2-ECD, have three regions (see Example 1) and/or three regions The upstream and downstream regions comprise hydrophilic amino acids (eg, G, T, S, N, E, and Y) and/or hydrophilic and potentially O/N glycosylation-modified amino acid substitutions. Specifically, the mutants of the present invention comprise T/S and N-X-S/T in the region (X can be any amino acid other than proline (P)), with hydrophilic enhancement and potential glycosylation modification protection as strategies, The stability of the fusion protein can be improved.

Compared with the fusion protein comprising wild-type TGFBR2-ECD, the fusion protein of the present invention comprises an improved TGFBR2-ECD mutant, so that the fusion protein of the present invention is less prone to breakage and degradation during production, and exhibits higher stability . TGFBR2-ECD mutants in fusion proteins of the invention are closer to wild-type TGFBR2-ECD than fusion proteins comprising truncated TGFBR2-ECD, possibly helping to reduce potential risk in an in vivo setting. Furthermore, the fusion protein of the present invention (eg mutant anti-PD-L1/TGF-β Trap) has higher affinity activity for TGF-β1 and PD-L1. Therefore, the TGFBR2-ECD mutant of the present invention and the fusion protein comprising the same are useful supplements for the druggability development of TGF-β Trap.

Example

A further understanding of the present invention may be obtained by reference to some specific examples given herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Obviously, various modifications and changes can be made to the present invention without departing from the essence of the present invention, therefore, these modifications and changes are also within the scope of protection claimed in this application.

Unless otherwise indicated, the reagents and apparatus used in the Examples section are commercially available.

Example 1 Construction and expression of anti-PD-L1/TGF-β Trap

Mass spectrometry showed that the breakpoint of wild-type anti-PD-L1/TGFBR2-ECD was mainly located at the N-terminus of TGFBR2-ECD (schematic structure is shown in Figure 1A), and its sequence was "IPPHV QKS V NNDMI VT DNNG AVKFPQL". It was verified by mass spectrometry that the degradation sites of wild-type TGFBR2-ECD were mainly located in three regions, namely the first region QKS and the second region N-NDM-I (where "-" represents the breakpoint), and the third region "DNNG" .

It can be seen from Figure 1A that the main degradation products produced by the fragmentation have a complete structure at the end of the antibody, and the degradation products cannot be effectively removed by conventional Protein A media, which brings great challenges to the development of downstream purification processes and seriously affects the development of molecular druggability. The inventors found that when the first region is mainly mutated (such as Mu1 and Mu3), the main degradation point will be transferred to the second region; and when only the first and second regions are modified (such as Mu2 and Mu4), the first The sequence "DNNG" of the three regions will become a new degradation site. Therefore, targeted and effective mutation optimization for three regions or three regions and their upstream and downstream sequences at the same time is an inevitable choice to obtain high-quality fusion proteins.

In this example, using anti-PD-L1 antibody as targeting moiety and TGFBR2-ECD wild type or mutant as TGF-β Trap, several bifunctional molecules containing TGF-β Trap and anti-PD-L1 antibody were constructed ( Anti-PD-L1/TGF-β Trap). The anti-PD-L1 antibody used in the examples is the B11 antibody described in WO 2019/233462, the relevant contents of which are incorporated herein by reference in their entirety. Anti-PD-L1 antibody B11 comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO:43 and two light chains each comprising the amino acid sequence of SEQ ID NO:42. The anti-PD-L1/TGF-β Trap contains two fusion polypeptides and two light chains, wherein the C-terminus of each antibody heavy chain (SEQ ID NO: 43) of antibody B11 is bound to a TGFBR2-ECD (wild-type or mutated). The N-terminus of anti-PD-L1/TGF-β Trap is connected by a linker (G ₄ S) ₄ G to form a fusion polypeptide, and the light chain of anti-PD-L1/TGF-β Trap is the light chain of antibody B11 (SEQ ID NO: 42).

Fusion protein Mu1-11 containing mutant TGFBR2-ECD, also known as mutant anti-PD-L1/TGF-β Trap. The TGFBR2-ECD mutants contained in Mu1-4 have the amino acid sequences of SEQ ID NOs: 44-47, respectively, and the fusion polypeptides have the amino acid sequences of SEQ ID NOs: 48-51, respectively. The TGFBR2-ECD mutants contained in Mu5-11 have the amino acid sequences of SEQ ID NOs: 2-8, respectively, and the fusion polypeptides of the TGFBR2-ECD mutants and the heavy chain of antibody B11 have the amino acid sequences of SEQ ID NOs: 9-15, respectively. Compared to Mu5-11, Mu1-4 contains amino acid mutations only in the first and/or second of the three regions (see Table 1).

A wild-type fusion protein (WT) comprising TGFBR2-ECD wild-type (SEQ ID NO: 1) was also constructed as a reference, also referred to as a wild-type anti-PD-L1/TGF-beta Trap (see Table 1).

It can be seen from the results in Table 2 that the simultaneous mutation transformation in the three regions or the three regions and their upstream and downstream regions is the key to improving the stability of the fusion protein. Mu5-9 are mutations targeting only three regions: Mu5 contains only one hydrophilic and potentially O-glycosylated amino acid "S" in each region; Mu6 contains two amino acids in each region Hydrophilic and potentially O-glycosylated amino acid "S/T"; Mu7, on the basis of Mu5, also contains a hydrophilic and potentially N-glycosylated amino acid "N" in the first region in the first region , and form a potential N-glycosylation site "NAS"; on the basis of Mu7, Mu8 also contains an amino acid "N" that is hydrophilic and can undergo potential N-glycosylation modification in the second region, and forms a potential N-glycosylation site "NAS"; on the basis of Mu8, Mu9 also contains a hydrophilic amino acid "N" that can undergo potential N-glycosylation modification in the third region, and forms a potential N-glycosyl The chemical site "NAS". Mu10 and Mu11 introduce a wide range of hydrophilic amino acids in the three regions and their upstream and downstream regions, especially the amino acid "S/T" that can undergo potential O-sugar modification. Table 1 Anti-PD-L1/TGF-β Trap fusion protein sequence structure TGFBR2-ECD fusion polypeptide WT Ab-linker-IPPHV QKS V NNDMI VT DNNG AVKF…NIIFSEEYNTSNPD SEQ ID NO: 1 unnumbered Mu1 Ab-linker-IPPHV Q G S V NNDMI VT DNNG AVKF…NIIFSEEYNTSNPD SEQ ID NO: 44 SEQ ID NO: 48 Mu2 Ab-linker-IPPHV Q G S V NND G I VT DNNG AVKF…NIIFSEEYNTSNPD SEQ ID NO: 45 SEQ ID NO: 49 Mu3 Ab-linker-IPPHV GG S V NND G I VT DNNG AVKF…NIIFSEEYNTSNPD SEQ ID NO: 46 SEQ ID NO: 50 Mu4 Ab-linker-IPPHV GG S V GGSG I VT DNNG AVKF…NIIFSEEYNTSNPD SEQ ID NO: 47 SEQ ID NO: 51 Mu5 Ab-linker-IPPHV GG S V GGSG I VT GGS G AVKF…NIIFSEEYNTSNPD SEQ ID NO: 2 SEQ ID NO: 9 Mu6 Ab-linker-IPPHV TG S V TGSG I VT TGS G AVKF…NIIFSEEYNTSNPD SEQ ID NO: 3 SEQ ID NO: 10 Mu7 Ab-linker-IPPHV NA S V GGSG I VT GGS G AVKF…NIIFSEEYNTSNPD SEQ ID NO: 4 SEQ ID NO: 11 Mu8 Ab-linker-IPPHV NA S V NASG IVT GGS G AVKF…NIIFSEEYNTSNPD SEQ ID NO: 5 SEQ ID NO: 12 Mu9 Ab-linker-IPPHV NA S V NASG I VT GNAS AVKF…NIIFSEEYNTSNPD SEQ ID NO: 6 SEQ ID NO: 13 Mu10 Ab-linker- T PPH T Q E S T SES MI T T GES G A T K Y ……NIIFSEEYNTSNPD SEQ ID NO: 7 SEQ ID NO: 14 Mu11 Ab-linker- T PPH T Q T S T NN S MI T T GTS G A T K Y …NIIFSEEYNTSNPD SEQ ID NO: 8 SEQ ID NO: 15 Note: Ab is an anti-PD-L1 antibody, which has two light chains and two heavy chains, the light chain has the amino acid sequence of SEQ ID NO: 42, and the heavy chain has the amino acid sequence of SEQ ID NO: 43; Bottom line is shown; linker is a linker (G ₄ S) ₄ G, through which the C-terminus of each heavy chain is linked to the N-terminus of a mutant to form a fusion polypeptide; amino acids contained in the TGFBR2-ECD mutant in Mu1-11 Mutations are shown in bold font.

A fusion protein Fc-TGFBR2-ECD (SEQ ID NO: 52) was also constructed, in which the N-terminus of the TGFBR2-ECD mutant (SEQ ID NO: 2) was linked to the C-terminus of the Fc fragment of human IgG1 (SEQ ID NO: 53) Connection via connector (G ₄ S) ₄ G.

Plasmids expressing wild-type and mutant anti-PD-L1/TGF-β Trap or fusion protein Fc-TGFBR2-ECD were transiently transfected into Expi-CHO cells (Thermo Fisher). After about 14 days of serum-free cell culture, the culture supernatant was harvested. The antibody expression in the culture supernatant was detected, and the culture supernatant was purified with Protein A medium to obtain the target fusion protein for subsequent detection.

Example 2 Stability detection of anti-PD-L1/TGF-β Trap

The wild-type and mutant anti-PD-L1/TGF-β Trap as described in Example 1 were purified by Protein A medium in one step, and then the content of degradants was detected by reduced protein electrophoresis, SEC-HPLC and CE-SDS.

Reduced protein electrophoresis: Take the sample and add an appropriate amount of Loading buffer, and add DTT to one of the samples for reduction. After the reduced sample is heated in a water bath at 70°C for 10 min, 2 μg of the sample is loaded and separated by 10% protein gel electrophoresis. The experimental results are shown in Figure 1B, lane 1 is anti-PD-L1 antibody, lane 2 is wild-type anti-PD-L1/TGF-β Trap, and lane 3 is Mu5. The results showed that the wild-type anti-PD-L1/TGF-β Trap had a significant fusion protein-HC degradation product near 53 kDa (TGFBR2-ECD deletion), and the degradation of Mu5 molecule was effectively controlled.

SEC-HPLC: 100 mM PB 200 mM Arg (pH 6.8) as mobile phase, 1 mL/min flow rate, analyzed on a Waters BEH SEC 200A 7.8x300 mm 3.5 μm column, 50 μg per sample injected.

CE-SDS: Take two samples and add an appropriate amount of sample buffer, one of which is added with β-ME (β-mercaptoethanol) for reduction, the non-reduced samples are water bathed at 70°C for 5 minutes, and the reduced samples are water bathed at 70°C for 10 minutes. CE-SDS analysis was performed using Maurice, with non-reducing samples (nrCE-SDS) separated for 40 min and reduced samples (rCE-SDS) separated for 35 min.

The profiles of the stability of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 analyzed by SEC-HPLC are shown in Figure 2: the proportion of monomers in WT was 85.2%, the proportion of degradants was 14%, and The monomer ratio in Mu5 is 97%.

Electropherograms of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 stability analyzed by nrCE-SDS are shown in Figure 3: the proportion of intact protein in WT was 86.4%, the proportion of protein fragments was as high as 13.6%, while The proportion of intact protein in Mu5 is as high as 94.7%, and the proportion of protein fragments is as low as 5.3%.

Electropherograms of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 stability analyzed by rCE-SDS are shown in Figure 4: The proportion of protein fragments in WT was as high as 8.3%, while the proportion of protein fragments in Mu5 was as low as 1.8%.

Table 2 summarizes the SEC-HPLC and CE-SDS data of wild-type (WT) and mutant anti-PD-L1/TGF-β Trap (Mu1-11).

The SEC-HPLC results in Table 2 show that the proportion of monomer (Mono) in the wild-type anti-PD-L1/TGF-β Trap is 85.2%, and the proportion of degradant (LMW) is as high as 14%. The proportion of monomers in L1/TGF-β Trap (Mu5-11) can reach more than 95%, and the proportion of degradants can be as low as 3.5%, indicating that the mutant anti-PD-L1/TGF-β Trap of the present invention is more stable. high.

The nrCE-SDS results in Table 2 show that the proportion of intact protein (Intact) in the wild-type anti-PD-L1/TGF-β Trap is 86.4%, and the proportion of protein fragments (Fragment) is as high as 13.6%. The proportion of intact protein in L1/TGF-β Trap (Mu5-11) was as high as 94.7% or more, and the proportion of protein fragments was as low as about 5%.

The rCE-SDS results in Table 2 show that the proportion of protein fragments of wild-type anti-PD-L1/TGF-β Trap is as high as 8.3%, and the protein fragments of mutant anti-PD-L1/TGF-β Trap (Mu5-11) of the present invention are as high as 8.3%. The proportion is as low as 2% or less. These results indicate that the mutant anti-PD-L1/TGF-β Trap of the present invention is more stable.

As can be seen from this table, the fragmentation-fragile regions (first region QKS (6-8 amino acids), second region NNDMI (10-14 amino acids) and third region of wild-type anti-PD-L1/TGF-β Trap Random mutations in the region DNNG (amino acids 17-20) do not necessarily have a significant quality improvement effect. Substitution of amino acids in these three regions with hydrophilic amino acids and/or N-X-S/T motifs can improve the anti-PD-L1/TGF-β Trap by comparing Mu5-11 with wild-type anti-PD-L1/TGF-β Trap. stability. Mu5, which was effectively mutated for the three regions at the same time, and Mu6-8, which continued to introduce hydrophilic and N-glycosylation-modified N-X-S/T motifs on this basis, had better quality improvement. In addition, Mu10/11 introduced a wide range of hydrophilic amino acids for the cleavable region and its nearby amino acid residues, especially the extensive introduction of amino acid T/S, which can undergo potential O-glycosylation modification, also significantly promoted anti-PD-L1. / TGF-β Trap stability.

In addition, compared with Mu5-11, which contains amino acid mutations in all three regions, Mu1-4 contains amino acid mutations in only one or two of the three regions, and such amino acid mutations are resistant to PD-L1/ The stability improvement of TGF-β Trap is not significant and may even aggravate protein degradation. It can be seen that to improve the stability of anti-PD-L1/TGF-β Trap, the amino acid mutation region also has a certain selectivity. Table 2 Quality detection of wild-type and mutant anti-PD-L1/TGF-β Trap

Example 3 Analysis of specific recognition activity of anti-PD-L1/TGF-β Trap

In this example, the binding of anti-PD-L1/TGF-β Trap to target proteins human PD-L1 (hPD-L1) and human TGF-β1 (hTGF-β1) was determined by ELISA.

Human PD-L1 (R&D Company) or TGF-β1 (Acro Company) were diluted with PBS to 1 μg/ml and coated on 96-well plates, respectively. After overnight incubation at 4°C, the coating solution was discarded the next day and washed with PBST. Then, 200 μl of BSA blocking solution was added to each well and incubated for 1 h at room temperature. Washed three times with PBST, wild-type anti-PD-L1/TGF-β Trap, anti-PD-L1/TGF-β Trap and PD-L1 antibodies of the present invention were diluted with 1% BSA PBST to a maximum concentration of 14 nM for 4-fold gradient dilution, 8 concentration gradients were made, and 100 μl per well was added to the 96-well plate as the primary antibody, and incubated at room temperature for 2 h. After washing three times with PBST, anti-human IgG-Fab-HRP (secondary antibody, Abcam) was added and incubated for 1 h at room temperature. Finally, each well was washed four times with PBST, and TMB color developing solution (Beijing Tiangen Biochemical Technology Co., Ltd.) was added to develop color at room temperature. After the color development was terminated, the OD value was measured with a microplate reader at 450 nm. The experimental results are shown in Figure 5. The EC50 of wild-type (WT) and mutant anti-PD-L1/TGF-β Trap (Mu5-11) binding to PD-L1 was between 0.1-0.2 nM (Figure 5A), and the The EC50 of TGF-β1 binding was between 0.1-0.2 nM (Fig. 5B), indicating that WT and Mu5-11 had similar in vitro binding abilities to the two target proteins PD-L1 and TGF-β1.

Example 4 Affinity analysis of anti-PD-L1/TGF-β Trap

In this example, the Octet RED96 detection system was used to determine the affinity of the fusion protein to the target protein TGF-β1.

Coupling ligand: This method adopts FAB2G (Anti-Human Fab-CH1 2nd Generation) wafer, soak the wafer in SD buffer for 10-15 min in advance, and load the ligand (wild-type or mutant anti-PD) at a concentration of 50 µg/ml -L1/TGF-β Trap), the preset duration is 1500 s, the preset value of ligand loading is 2.5 nm, and the wafer is infiltrated with running buffer (SD buffer) until the baseline is stable, and free ligands are removed.

Kinetic experiments: TGF-β1 was diluted with SD buffer to 44.4 nM, 22.2 nM, 11.1 nM, 5.55 nM, 2.775 nM and 1.39 nM, respectively. Using TGF-β1 as the analyte and wild-type and mutant anti-PD-L1/TGF-β Trap as the ligand, the affinity detection experiment was performed. The ligand-analyte binding time (Association) was 240 s, the analyte dissociation time (Dissociation) was 300 s, and SD buffer was used as a blank control for background subtraction.

Forté Bio Data Analysis 9.0 was used for analysis using a static fitting model. The results are shown in Figure 6. The KD between wild-type anti- _PD -L1/TGF-β Trap (WT) and TGF-β1 was 7.5×10 ^-9 M , the K _D between Mu5 and TGF-β1 was 3.6×10 ^-9 M, indicating that WT and Mu5 had similar binding affinities to TGF-β1.

Example 5 Expression regulation of EMT pathway genes by anti-PD-L1/TGF-β Trap

A549 human non-small cell lung cancer cells (Cell Bank of Chinese Academy of Sciences, SCSP-503) were inoculated in 6-well plates and cultured for 24-36 h to 60-70% confluency, the control group was added with 2 ng/ml TGF-β1 F-12K complete medium, the experimental group was added 5 μg/ml wild-type anti-PD-L1/TGF-β Trap or Mu5 to the complete medium containing TGF-β1, respectively.

After co-cultivation for 48 h, the cells were recovered, and total cellular RNA was extracted, and cDNA was obtained by reverse transcription reaction. A real-time fluorescent PCR reaction system was prepared to detect whether anti-PD-L1/TGF-β Trap was involved in inhibiting the key genes of the downstream EMT (epithelial-mesenchymal transition) pathway induced by TGF-β. The key genes of related pathways mainly include CDH2, MMP-2, MMP-3, MMP-9, TWIST2 and Vimentin. The results are shown in FIG. 7 , both the wild-type anti-PD-L1/TGF-β Trap (WT) and the anti-PD-L1/TGF-β Trap (Mu5) of the present invention can effectively inhibit TGF-β1-induced EMT pathway gene expression .

Example 6 Validation of the efficacy of Mu5 on subcutaneous transplanted tumor of colon cancer cells in MC38/hPD-L1 mice

In this example, Mu5 was used as a representative drug of the anti-PD-L1/TGF-β Trap fusion protein of the present invention to verify the efficacy of transplanted tumors. 1×10 ⁶ colon cancer MC38/hPD-L1 (human PD-L1 stably transfected MC38 cell line; constructed by the Animal Experiment Center) cells were injected into the armpits of mice. After the tumors grew to an average volume of about 50 mm ³ , the mice were randomly divided into 3 groups according to the tumor volume: the solvent control group (PBS), the Mu5 1.22 mg/kg group and the Mu5 3.66 mg/kg group. There were 8 animals in each group. Each group was given the corresponding concentration of the test substance by intraperitoneal injection at a dose of 10 ml/kg. The dosing frequency was twice a week, and the tumor volume was weighed and measured twice a week. The initial dose was Day 1 (D1). Mice were weighed and tumor volume (TV) was measured on days 1, 4, 7, 11, 15, and 18 (D18), respectively. On day 18, mice were sacrificed, and tumors were dissected and weighed (Tumor Weight, TW). Relative tumor volume (RTV), relative tumor proliferation rate (T/C) and tumor weight inhibition rate (Inhibition Ratio, IR) were calculated according to tumor volume and tumor weight. The results are shown in Table 3, Table 4 and FIG. 8 .

) RTV (

) T/C( ％ ) D1 D18 PBS - 52±3 2215±226 41.96±2.89 - Mu5 1.22 49±2 849±129 ^*** 17.45±2.81 ^*** 41.60 Mu5 3.66 50±2 606±168 ^*** 12.27±3.42 ^*** 29.25 注：TV = 0.5×a×b ²，其中a、b分別表示腫瘤的長和寬。RTV = Vt/V0，其中V0為分組給藥時(即D1)所測得的腫瘤體積，Vt為每一次測量時的腫瘤體積。T/C= (T _RTV/ C _RTV)×100%，其中T _RTV為治療組的RTV，C _RTV為溶劑對照組的RTV。與PBS組相比， ^***： P＜0.001。

，平均值±標準誤差。 As shown in Table 3 and Figure 8, on day 18, the RTV values of the Mu5 1.22 mg/kg group and the Mu5 3.66 mg/kg group were 17.45±2.89, respectively, compared to the RTV value of the solvent control group (PBS) of 41.96±2.89, respectively 2.81 and 12.27 ± 3.42. Table 3 Tumor inhibitory effect of Mu5 (tumor volume) group Dosage (mg/kg) TV( mm ³ ) (

) RTV (

) T/C( % ) D1 D18 PBS - 52±3 2215±226 41.96±2.89 - Mu5 1.22 49±2 849±129 ^*** 17.45±2.81 ^*** 41.60 Mu5 3.66 50±2 606±168 ^*** 12.27±3.42 ^*** 29.25 Note: TV = 0.5×a×b ² , where a and b represent the length and width of the tumor, respectively. RTV = Vt/V0, where V0 is the tumor volume measured at the time of group administration (ie, D1), and Vt is the tumor volume at each measurement. T/C=(T _RTV / C _RTV )×100%, where T _RTV is the RTV of the treatment group, and C _RTV is the RTV of the solvent control group. ^*** : P < 0.001 compared to the PBS group.

, mean ± standard error.

) IR( ％ ) PBS - 2.4269±0.3936 - Mu5 1.22 1.1161±0.0856 ^** 54.01 Mu5 3.66 0.6751±0.2231 ^{* *} 72.18 注：IR = (1- TW _T/TW _C)×100%，其中TW _T為治療組的瘤重，TW _C為對照組的瘤重。與PBS組相比， ^**： P＜0.01。

，平均值±標準誤差。 As shown in Table 4, on day 18, the tumor weights of the Mu5 1.22 mg/kg and Mu5 3.66 mg/kg groups were 1.1161 ± 0.0856 g, respectively, compared with the solvent control (PBS) tumor weight of 2.4269 ± 0.3936 g, respectively and 0.6751 ± 0.2231 g. The tumor weight inhibition rate (IR) of Mu5 1.22 mg/kg group and Mu5 3.66 mg/kg group were 54.01% and 72.18%, respectively. Table 4 Tumor inhibitory effect of Mu5 (tumor weight) group Dosage (mg/kg) Tumor weight (g) (

) IR( % ) PBS - 2.4269±0.3936 - Mu5 1.22 1.1161±0.0856 ^** 54.01 Mu5 3.66 0.6751±0.2231 ^** 72.18 Note: IR = (1-TW _T /TW _C )×100%, where _{T T} is the tumor weight in the treatment group, and T _C is the tumor weight in the control group. ^** : P < 0.01 compared to the PBS group.

, mean ± standard error.

As shown in Table 3, Table 4 and Figure 8, compared with the solvent control group, tumor growth was significantly inhibited in the Mu5 treatment group, and showed a good dose-dependent effect. sequence listing SEQ ID NO: 1 Wild-type TGF-βRII extracellular region 1 IPPHVQKSVN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 2 TGF-βRII extracellular region of Mu5 1 IPPHVGGSVG GSGIVTGGSG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 3 TGF-βRII extracellular region of Mu6 1 IPPHVTGSVT GSGIVTTGSG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 4 TGF-βRII extracellular region of Mu7 1 IPPHVNASVG GSGIVTGGSG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 5 TGF-βRII extracellular region of Mu8 1 IPPHVNASVN ASGIVTGGSG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 6 TGF-βRII extracellular region of Mu9 1 IPPHVNASVN ASGIVTGNAS AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 7 TGF-βRII extracellular region of Mu10 1 TPPHTQESTS ESMITTGESG ATKYPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 8 TGF-βRII extracellular region of Mu11 1 TPPHTQTSTN NSMITTGTSG ATKYPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 9 Fusion polypeptide of Mu5 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVGGSVGGS 481 GIVTGGSGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 10 Fusion polypeptide of Mu6 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVTGSVTGS 481 GIVTTGSGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 11 Fusion polypeptide of Mu7 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVNASVGGS 481 GIVTGGSGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 12 Fusion polypeptide of Mu8 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVNASVNAS 481 GIVTGGSGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 13 Fusion polypeptide of Mu9 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVNASVNAS 481 GIVTGNASAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 14 Fusion polypeptide of Mu10 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG TP PHTQESTSES 481 MITTGESG ATKYPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 15 Fusion polypeptide of Mu11 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG TP PHTQTSTNNS 481 MITTGTSG ATKYPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 16 B10#HCDR1 DSWIH SEQ ID NO: 17 B10#HCDR2 WISPFGGSTYYADSVKG SEQ ID NO: 18 B10#HCDR3 RHWPGGFDY SEQ ID NO: 19B10#VH 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSS SEQ ID NO: 20B10#LCDR1 RASQDVSTAVA SEQ ID NO: 21B10#LCDR2 SASFLYS SEQ ID NO: 22B10#LCDR3 QQFIFHPAT SEQ ID NO: 23 B10#VL 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FIFHPATFGQ GTKVEIK SEQ ID NO: 24 B11#HCDR1 ETWLH SEQ ID NO: 25 B11#HCDR2 WVSPFGGSTYYADSVKG SEQ ID NO: 26 B11#HCDR3 RHWPGGFDY SEQ ID NO: 27 B11#VH 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSS SEQ ID NO: 28 B11#LCDR1 RASQDVSTAVA SEQ ID NO: 29B11#LCDR2 SASFLYS SEQ ID NO: 30 B11#LCDR3 QQFLYHPAT SEQ ID NO: 31 B11#VL 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FLYHPATFGQ GTKVEIK SEQ ID NO: 32 H12#HCDR1 ESWLH SEQ ID NO: 33 H12#HCDR2 WVTPYGGSTYYADSVKG SEQ ID NO: 34 H12#HCDR3 RHWPGGFDY SEQ ID NO: 35 H12#VH 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ESWLHWVRQA PGKGLEWVAW VTPYGGSTYY 61 ADSVKGRFTI SADTSKNTAY LMNSLRAEDT AVYYCARRHW PGGFDYWGQG TLVTVSS SEQ ID NO: 36 H12#LCDR1 RASQDVSTAVA SEQ ID NO: 37 H12#LCDR2 SASFLYS SEQ ID NO: 38H12#LCDR3 QQFLYHPAT SEQ ID NO: 39 H12#VL 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 61 RFSGSGSGTD FTLTISSLPE DFATYYCQQF LYHPATFGQG TKVEIK SEQ ID NO: 40 light chain constant region 1 RTVAAPSVFF PPSDELKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK 61 DSTYSLSSTL TLSKADYEKH KVYACEVTHG LSSPVTKSFN RGEC SEQ ID NO: 41 Heavy chain constant region 1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 61 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 121 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYA 181 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE 241 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPG SEQ ID NO: 42 B11# light chain 1 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 61 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FLYHPATFGQ GTKVEIKRTV AAPSVFIFPP 121 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTVDY 181 V T E K S K SP LSKA SEQ ID NO: 43 B11# heavy chain 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG SEQ ID NO: 44 TGF-βRII extracellular region of Mu1 1 IPPHVQ G SVN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 45 TGF-βRII extracellular region of Mu2 1 IPPHVQ G SVN ND G IVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 46 TGF-βRII extracellular region of Mu3 1 IPPHV GG SVN ND G IVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 47 TGF-βRII extracellular region of Mu4 1 IPPHV GG SV G GSG IVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV 61 CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC 121 NDNIIFSEEY NTSNPD SEQ ID NO: 48 Fusion polypeptide of Mu1 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVQ G SVNND 481 MIVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 49 Fusion polypeptide of Mu2 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHVQ G SVNND 481 G IVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 50 Fusion polypeptide of Mu3 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHV GG SVNND 481 G IVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 51 Fusion polypeptide of Mu4 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS ETWLHWVRQA PGKGLEWVAW VSPFGGSTYY 61 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 121 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 181 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 241 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 361 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 421 GNVFSCSVMH EALHNHYTQK SLSLSPG GGG GSGGGGSGGG GSGGGGSG IP PHV GG SV GGS 481 G IVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI 541 TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSSDECND NIIFSEEYNT 601 SNPD SEQ ID NO: 52 Fc-TGFBR2-ECD 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYASTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGGGGG SGGGGSGGGG 241 SGGGGSGIPP HVGGSVGGSG IVTGGSGAVK FPQLCKFCDV RFSTCDNQKS CMSNCSITSI 301 CEKPQEVCVA VWRKNDENIT LETVCHDPKL PYHDFILEDA ASPKCIMKEK KKPGETFFMC 361 SCSSDECNDN IIFSEEYNTS NPD SEQ ID NO: 53 Human IgG1 Fc fragment 1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYASTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG

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Figure 1A: Schematic diagram of the structure of anti-PD-L1/TGF-β Trap and the degradation of wild-type anti-PD-L1/TGF-β Trap. Figure 1B: Reduced protein electrophoresis of wild-type anti-PD-L1/TGF-β Trap and mutant anti-PD-L1/TGF-β Trap, and the protein bands were visualized by silver staining: lane 1 is anti-PD-L1 antibody , lane 2 is wild-type anti-PD-L1/TGF-β Trap, lane 3 is Mu5; HC, heavy chain; LC, light chain; fusion protein-HC, fusion of anti-PD-L1 antibody heavy chain and TGFBR2-ECD Polypeptide; fusion protein-LC, antibody light chain in anti-PD-L1/TGF-β Trap, identical to anti-PD-L1 antibody light chain; degradation product, fusion protein-HC due to N-terminal cleavage of TGFBR2-ECD Deletion of the TGFBR2-ECD product. Figure 2: SEC-HPLC profiles showing the proportions of components in wild-type anti-PD-L1/TGF-β Trap (WT) (2A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (2B) ; Mono, monomer; LMW, low molecular weight degradant. Figure 3: Non-reducing CE-SDS capillary electrophoresis patterns showing wild-type anti-PD-L1/TGF-β Trap (WT) (3A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (3B) The ratio of each component: Fragment, incomplete structural protein; Intact, complete protein. Figure 4: Reduced CE-SDS capillary electrophoresis patterns showing wild-type anti-PD-L1/TGF-β Trap (WT) (4A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (4B) Ratio of components: HC, antibody heavy chain in anti-PD-L1/TGF-β Trap and fusion polypeptide of TGFBR2-ECD; LC, antibody light chain in anti-PD-L1/TGF-β Trap; Fragment, incomplete Structural protein fragment; Intact, intact protein. Figure 5: Binding of wild-type anti-PD-L1/TGF-β Trap (WT) and mutant anti-PD-L1/TGF-β Trap (Mu5-11) to hPD-L1 (5A) and hTGF-β1 (5B) Active ELISA test results. Figure 6: Molecular dynamics assay results of the interaction of wild-type anti-PD-L1/TGF-β Trap (WT) (6A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (6B) with hTGF-β1 . Figure 7: The results of detecting the expression inhibition of EMT pathway genes by wild-type anti-PD-L1/TGF-β Trap and mutant anti-PD-L1/TGF-β Trap in A549 cell line: in the presence of hTGF-β1, respectively The expression inhibition of EMT pathway genes by wild-type anti-PD-L1/TGF-β Trap and mutant PD-L1/TGF-β Trap was detected. The detected genes were from left to right: CDH2, MMP2, MMP3, MMP9, TWIST1, VIMENTIN; cell samples are: (1) TGF-b1 2 ng: hTGF-β1 with a final concentration of 2 ng/ml was added to the medium, cultured for 48 h, as a negative control; (2) TGF-b1+WT: TGF -b1 2 ng/ml, at the same time adding 5 μg/ml wild-type PD-L1/TGF-β Trap, cultured for 48h; (3) TGF-b1+Mu5: TGF-b1 2 ng/ml, at the same time adding 5 μg/ml Mu5, cultured for 48h. Figure 8: In vivo tumor-suppressive activity of mutant anti-PD-L1/TGF-β Trap (Mu5) detected in MC38/hPD-L1 mouse colon cancer cell subcutaneous xenograft model; the curves show in each group of mice over time relative tumor volume.

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Claims (31)

A mutant of the extracellular region of TGF-βRII capable of binding TGF-β and carrying one or more amino acid mutations at the N-terminus, such that the mutant is contained in the extracellular region of TGF-βRII compared to the wild-type TGF-βRII Amino acid mutation and/or improvement of hydrophilicity in the breakable region. The mutant as claimed in claim 1, which comprises the amino acid sequence of SEQ ID NO: 1, and has one or more amino acid mutations in its N-terminal amino acids 1-24; Preferably, the mutant comprises amino acid mutations in each of the three regions and at least one S or T in each of the three regions, The three regions include: the first region QKS (amino acids 6-8), the second region NNDMI (amino acids 10-14) and the third region DNNG (amino acids 17-20). A mutant as claimed in claim 1 or 2, wherein The amino acid mutation comprises amino acid substitution in each of the three regions with a hydrophilic amino acid, an N-X-S/T motif, or a combination thereof, wherein X is any amino acid other than proline (P); Preferably, at least one substitution is made in each of the three regions; More preferably, at least two substitutions are made in each of the three regions. The mutant of claim 3, wherein The hydrophilic amino acid is selected from G, T, S, N, E, Y or a combination thereof; The N-X-S/T motif is N-A-S. The mutant of any one of claims 1-4, wherein the amino acid mutation comprises Mutation of the first region QKS to GGS, TGS or NAS, mutating the second region NNDMI to GGSGI, TGSGI, or NASGI, and Mutating the third region DNNG to GGSG, TGSG or GNAS; Preferably, the mutant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6. The mutant of claim 1 or 2, further comprising amino acid mutations in the upstream and downstream regions of the three regions; Preferably, the amino acid mutation comprises substituting at least one amino acid in each of the three regions with G, S, E, T or a combination thereof, and substituting T, Y or a combination in the upstream and downstream regions of each region at least one amino acid is substituted in; More preferably, the amino acid mutation includes mutating the first amino acid I to T, 5-8 amino acids (VQKS) to TQES or TQTS, and 9-15 amino acids (VNNDMIV) to TSESMIT or TNNSMIT. , and mutating amino acids 17-24 (DNNGAVKF) to GESGATKY or GTSGATKY; Most preferably, the mutant has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. A targeting molecule comprising the mutant of any one of claims 1-6 and a targeting moiety, wherein the targeting moiety is covalently linked to the N-terminus of the mutant; Preferably, the molecules targeted by the targeting moiety are selected from the group consisting of growth factors and their receptors, cytokines and their receptors, cell surface antigens, immune checkpoint molecules, hormones and other biologically active components in vivo. The targeting molecule of claim 7, wherein the molecule targeted by the targeting moiety is selected from the group consisting of CD13, CD19, CD22, CD25, CD27, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352, CLDN18.2, DLL3, CN33, GPNMB, ENPP3, Nectin-4, HER2/ErbB2, EGFR, FGFR, VEGFR, HGFR, PDGFR, VEGF, HGF, FGF, FGF-2, PDGF, Galectin-3, EGFSLC44A4/AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, SLITRK6, SLAMF7/CS1, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, CA6, Integrin αV, TDGF1, Ephrin A4, PTK7, NOTCH3, C4.4A, FLT3, PD1, CTLA4, LAG-3, BTLA, TIM-2 , LAIR1, PD-L1, B7-DC, B7-H3, B7-H4, HVEM and TIM-4 group. The targeting molecule of claim 7 or 8, wherein the targeting moiety is an Fc fragment, an antibody or antigen-binding fragment thereof, a mimetibody, a ligand, a ligand-binding domain or a functionally active domain of an in vivo active protein ; Preferably, the ligands are cell surface antigens, cytokines, growth factors or hormones; the in vivo active proteins are receptors, kinases, ion channels or other proteins involved in signal transduction; More preferably, the ligand binding domain or functional domain is a ligand binding domain or functional domain of a T cell receptor, cytokine receptor, growth factor receptor or hormone receptor; In particular, the targeting moiety is an antibody or antigen-binding fragment thereof that targets the tumor microenvironment. The targeting molecule of any one of claims 7-9, wherein the cytokine is selected from the group consisting of: G-CSF, GM-CSF, eotaxin/CCL11, MCP-1/CCL2, MIP-1α/CCL4, RANTES /CCL5, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17 , IL-18, IL-23, INFα, INFβ, INFγ and TNFα and TNFβ; the growth factor is selected from: VEGF, HGF, FGF, FGF-2, PDGF, IGF, TGF, NGF, EPO and EGF; the The hormones are peptide hormones and steroid hormones, preferably peptide hormones. The targeting molecule of any one of claims 7-10, wherein the targeting moiety is selected from the group consisting of synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, non-human antibodies, Humanized Antibodies, Chimeric Antibodies, Intrabodies, Single Domain Antibodies, Fab Fragments, Fab' Fragments, F(ab') ₂ Fragments, Fv Fragments, dsFv Fragments, Fd Fragments, Fd' Fragments, scFv, scFab and Bis group of antibodies. The targeting molecule of any one of claims 7-11, wherein the targeting moiety is an anti-PD-L1 antibody or an antigen-binding fragment thereof. A fusion polypeptide comprising the mutant of any one of claims 1-6 and a first polypeptide, wherein the C-terminus of the first polypeptide is covalently linked to the N-terminus of the mutant. The fusion polypeptide of claim 13, wherein the first polypeptide comprises at least one heavy chain variable region of an antibody or at least one light chain variable region of an antibody. The fusion polypeptide of claim 14, wherein the antibody is an anti-PD-L1 antibody. The fusion polypeptide according to any one of claims 13-15, further comprising a linker between the first polypeptide and the mutant, preferably, the linker is (G ₄ S) _n G, wherein n is an integer of 2-6, preferably an integer of 3-5, more preferably 4. A fusion protein comprising the fusion polypeptide of any one of claims 13-16 and a second polypeptide, wherein the second polypeptide, when combined with the first polypeptide, forms a targeting moiety, the targeting Parts are as defined in any of claims 7-12. The fusion protein of claim 17, wherein The first polypeptide comprises at least one heavy chain variable region of an antibody, and the second polypeptide comprises at least one light chain variable region of an antibody; or The first polypeptide comprises at least one light chain variable region of an antibody and the second polypeptide comprises at least one heavy chain variable region of an antibody. The fusion protein of claim 18, wherein the antibody is an anti-PD-L1 antibody. The fusion protein of claim 19, comprising: (1-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (1-2) A second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (2-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, HCDR3 comprising the amino acid sequence of SEQ ID NO: 26; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2-2) A second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 28, LCDR2, which comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or (3-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 34; and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (3-2) A second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO: 36, LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence of SEQ ID NO:38. The fusion protein of claim 19, comprising: (1-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (1-2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23; or (2-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2-2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31; or (3-1) Fusion polypeptide, from N-terminus to C-terminus, including: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (3-2) A second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39. The fusion protein of claim 19, comprising: (1) Fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and (ii) a mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8; and (2) a second polypeptide comprising a light chain comprising the amino acid sequence of SEQ ID NO:42. The fusion protein of any one of claims 17-22, further comprising: a linker between the first polypeptide and the mutant, preferably, the linker is (G ₄ S) _n G , wherein n is an integer of 2-6, preferably an integer of 3-5, more preferably 4. The fusion protein of any one of claims 17-23, which forms a dimer. A pharmaceutical composition, comprising the targeting molecule of any one of claims 7-12, the fusion polypeptide of any one of claims 13-16, or the fusion protein of any one of claims 17-24, and a pharmaceutically acceptable accepted carrier. A targeting molecule according to any one of claims 7-12, a fusion polypeptide according to any one of claims 13-16, a fusion protein according to any one of claims 17-24, or the pharmaceutical composition of claim 25 in preparation Use in medicaments for the treatment and/or prevention of neoplastic diseases; Preferably, the neoplastic disease is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, Nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, Endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or Ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancer, asbestos-induced cancer, and combinations thereof; More preferably, the neoplastic disease is lung cancer, ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer Cancer, laryngeal cancer, cervical cancer, endometrial cancer, osteosarcoma, colorectal cancer and non-small cell lung cancer, metastatic cancer, most preferably, the neoplastic disease is PD-L1 expressing metastatic cancer. A use of the fusion protein of any one of claims 19-24 in the preparation of a medicament for the treatment of cancer; preferably, the cancer is PD-L1 positive cancer; more preferably, the cancer is selected from lung cancer, Ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, endometrial cancer, Osteosarcoma, colorectal cancer, and non-small cell lung cancer. A nucleic acid molecule encoding the mutant of any one of claims 1-6, the targeting molecule of any one of claims 7-12, the fusion polypeptide of any one of claims 13-16, or The fusion protein of any one of claims 17-24. An expression vector comprising the nucleic acid molecule of claim 28. A host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29. A method for producing a mutant of any one of claims 1-6, a targeting molecule of any one of claims 7-12, a fusion polypeptide of any one of claims 13-16, or claim 17- The fusion protein of any one of 24, the method comprising: (i) culturing the host cell of claim 30 under conditions suitable for expression of the nucleic acid molecule or expression vector; and (ii) isolating and purifying the mutant, targeting molecule, fusion polypeptide or fusion protein.

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