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WO2013164325A1 - Multispecific antigen binding proteins - Google Patents

Multispecific antigen binding proteins Download PDF

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Publication number
WO2013164325A1
WO2013164325A1 PCT/EP2013/058946 EP2013058946W WO2013164325A1 WO 2013164325 A1 WO2013164325 A1 WO 2013164325A1 EP 2013058946 W EP2013058946 W EP 2013058946W WO 2013164325 A1 WO2013164325 A1 WO 2013164325A1
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WO
WIPO (PCT)
Prior art keywords
antigen binding
antibody
domain
binding protein
heavy chain
Prior art date
Application number
PCT/EP2013/058946
Other languages
French (fr)
Inventor
Sabine Imhof-Jung
Hubert Kettenberger
Christian Klein
Joerg Thomas Regula
Wolfgang Schaefer
Juergen Michael Schanzer
Original Assignee
F. Hoffmann-La Roche Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F. Hoffmann-La Roche Ag filed Critical F. Hoffmann-La Roche Ag
Publication of WO2013164325A1 publication Critical patent/WO2013164325A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to multispecific antibody, especially to a bispecific, bivalent antibody (comprising as one antigen binding site a monovalent antigen binding protein), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
  • US 2004/0033561 describes a method for the generation of monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL- CH2-CH3 antibody chain; however, a disadvantage of this method is the formation of a binding inactive homodimer of VL-CL-CH2-CH3 chains. Due the similar molecular weight such homodimeric by-products are the difficult to separate.
  • WO 2007/048037 also refers to monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain, but having a tagging moiety attached to the heavy chain for easier purification of the heterodimer from the difficult-to-separate homodimeric by-product.
  • WO 2009/089004 describes another possibility to generate a heterodimeric monovalent antibody using electrostatic steering effects.
  • the invention comprises a multispecific antigen binding protein, comprising
  • A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
  • the multispecific antigen binding protein according to the invention is a bispecific, bivalent antibody.
  • the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and
  • the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a Fv fragment, Fab fragment, a scFv fragment, and a scFab fragment. In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a Fv fragment and Fab fragment which are fused via two peptide connectors to the C-terminus or N-terminus of both heavy chains under A).
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a scFv fragment and scFab fragment which are fused via one peptide connector to the C-terminus or N-terminus of both heavy chains under
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a Fv fragment.
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a Fab fragment.
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a scFv fragment.
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a scFab fragment.
  • B) is fused via one or two peptide connectors to the N-terminus of one or both heavy chains under A).
  • the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus of one or both heavy chains under A).
  • the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and b) the amino acid sequence of SEQ ID NO:9;
  • the multispecific antigen binding protein according to the invention is characterized in that the heavy chains under A) are of human IgG isotype.
  • the multispecific antigen binding protein according to the invention is characterized in that the heavy chains under A) are of human IgGl or IgG4 isotype.
  • the invention further comprises a method for the preparation of a multispecific antigen binding protein according to the invention comprising the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding
  • a monovalent antigen binding protein according to the invention b) culturing the host cell under conditions that allow synthesis of said multispecific antigen binding protein molecule;
  • the invention further comprises nucleic acid encoding the multispecific antigen binding protein according to the invention.
  • the invention further comprises vectors comprising nucleic acid encoding the multispecific antigen binding protein according to the invention.
  • the invention further comprises host cell comprising said vectors.
  • the invention further comprises composition, preferably a pharmaceutical or a diagnostic composition of a multispecific antigen binding protein according to the invention.
  • the invention further comprises pharmaceutical composition comprising a multispecific antigen binding protein according to the invention and at least one pharmaceutically acceptable excipient.
  • the invention further comprises method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of a multispecific antigen binding protein binding protein according to the invention.
  • the multispecific antigen binding proteins according to the invention have valuable properties like low aggregation tendency, high stability, valuable pharmacokinetic properties (like e.g. halftime (term tl/2) or AUC) and are producible in good yields.
  • the multispecific antigen binding proteins according to the invention have valuable characteristics such as biological or pharmacological activities (as e.g. ADCC, or antagonistic biological activity as well as lack of agonistic activities). They can be used e.g. for the treatment of diseases such as cancer.
  • Figure 1 A - H) Scheme of different multispecific antigen binding protein according to the invention comprising a monovalent antigen binding protein (under A) i)) with VL-CL-CH2-CH3 chain and VH-CH1-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen.
  • a monovalent antigen binding protein under A) i)
  • VL-CL-CH2-CH3 chain and VH-CH1-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen.
  • FIG. 2 A - H) Scheme of different multispecific antigen binding protein according to the invention comprising a monovalent antigen binding protein (under A) ii)) with VL-CH1-CH2-CH3 chain and VH-CL-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen.
  • a monovalent antigen binding protein under A) ii)
  • VL-CH1-CH2-CH3 chain and VH-CL-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen.
  • an antigen binding peptide scFv, scFab, Fv or Fab
  • the invention comprises a multispecific antigen binding protein, comprising
  • A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody;
  • the multispecific antigen binding protein according to the invention is a bispecific, bivalent antibody.
  • the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody.
  • the multispecific antigen binding protein according to the invention is characterized in that the a monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
  • the CH3 domains of said monovalent antigen binding protein under A can be altered by the "knobs-into-holes" (KiH) technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al, Protein Eng. 9 (1996) 617-621; and
  • said monovalent antigen binding protein under A), which specifically binds to a first antigen is further characterized in that the CH3 domain of the heavy chain of the full length antibody of a) and the CH3 domain of the modified heavy chain of the full length antibody of b) each meet at an interface which comprises an original interface between the antibody CH3 domains;
  • said interface is altered to promote the formation of the monovalent antigen binding protein, wherein the alteration is characterized in that: i) the CH3 domain of one heavy chain is altered,
  • an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and
  • an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.
  • amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
  • amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).
  • both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.
  • C cysteine
  • said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises a T366W mutation in the CH3 domain of the "knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain”.
  • CH3 domains can also be used (Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the "knobs chain” and a E356C mutation or a S354C mutation into the CH3 domain of the "hole chain”.
  • said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C,
  • T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering always according to EU index of Kabat).
  • knobs-in-holes technologies as described by EP 1 870 459 Al, can be used alternatively or additionally.
  • a preferred example for said monovalent antigen binding protein are R409D; K370E mutations in the CH3 domain of the "knobs chain” and D399K; E357K mutations in the CH3 domain of the "hole chain” (numbering always according to EU index of Kabat).
  • said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises a T366W mutation in the CH3 domain of the "knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain” and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain” and D399K; E357K mutations in the CH3 domain of the "hole chain”.
  • said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain” and D399K; E357K mutations in the CH3 domain of the "hole chain”.
  • the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and
  • the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 10; and
  • the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 12;
  • the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 14;
  • full length antibody denotes a full length antibody consisting of two antibody heavy chains and two antibody light chains (see Fig. 1).
  • a heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH 1 -HR-CH2-CH3 ; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE.
  • VH antibody heavy chain variable domain
  • CHI antibody constant heavy chain domain 1
  • HR antibody hinge region
  • CH2 antibody heavy chain constant domain 2
  • CH3 antibody heavy chain constant domain 3
  • the heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of VH, CHI, HR, CH2 and CH3.
  • the light chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL.
  • the antibody light chain constant domain (CL) can be ⁇ (kappa) or ⁇ (lambda).
  • the antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHI domain (i.e. between the light and heavy chain) and between the hinge regions of the full length antibody heavy chains.
  • full length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE.)
  • Such full length antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies.
  • the full length antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same (first) antigen.
  • the monovalent antigen binding protein under A), which specifically binds to a first antigen are derived by modifying: a) the first heavy chain of said antibody by replacing the VH domain by the VL domain of said antibody; and by modifying b) the second heavy chain of said antibody by replacing the CHI domain by the CL domain of said antibody.
  • the monovalent antigen binding protein under A), which specifically binds to a first antigen comprise two modified heavy chains and no light chains.
  • antigen binding peptide refers to a monovalent antigen binding fragment or derivative of a full length antibody which includes an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), or a pair of VH/VL derived from full length antibodies or antibody fragments such as a VH domain and/or a VL domain, a Fv fragment, a single chain Fv (scFv) fragment, a Fab fragment, or single chain Fab (scFab) fragment.
  • the antigen binding peptide is a single variable heavy chain domain (sVH) with sufficient binding affinity to specifically bind to the respective antigen.
  • the antigen binding peptide comprises at least an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL).
  • such antigen binding peptides are selected from the group consisting of a VH domain, a single chain Fv (scFv) fragment, and a single chain Fab (scFab) fragment, preferably from the group consisting of Fv fragment, a Fab fragment, a single chain Fv (scFv) fragment and a single chain Fab (scFab) fragment.
  • the term "peptide connector" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin.
  • peptide connectors according to invention are used to fuse the antigen binding peptides to the C-or N-terminus of the full length and/or modified full length antibody chains to form a trispecific or tetraspecific antibody according to the invention.
  • said peptide connectors under c) are peptides with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids.
  • said peptide connector is
  • the C-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the C-terminus of said heavy or light chain.
  • binding site or "antigen-binding site” as used herein denotes the region(s) of antigen binding protein according to the invention to which a ligand (e.g the antigen or antigen fragment of it) actually binds and which is derived from antibody molecule or a fragment thereof (e.g. a Fab fragment).
  • the antigen-binding site according to the invention comprise an antibody heavy chain variable domains (VH) and an antibody light chain variable domains (VL).
  • VH antibody heavy chain variable domains
  • VL antibody light chain variable domains
  • An antigen-binding site of a monovalent antigen binding protein of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRLl, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.
  • Antibody or antigen binding protein specificity refers to selective recognition of the antibody or antigen binding protein for a particular epitope of an antigen.
  • Natural antibodies for example, are monospecific.
  • Multispecific antigen binding proteins have more than one antigen-binding specificities and are e.g. bispecific , tri- or tetraspecific. In one embodiment of the invention they are bispecific, in one embodiment they are bispecific, bivalent.
  • Bispecific antigen binding proteins have two different antigen-binding specificities. Trispecific antigen binding proteins accordingly have three different antigen-binding specificities. Tetraspecific antigen binding proteins have four different antigen-binding specificities.
  • the term "valent" as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule or antigen binding protein.
  • a natural antibody for example or a full length antibody according to the invention has two binding sites and is bivalent.
  • the term monovalent refers to one binding site in an antigen binding protein, therefore to only one specificity.
  • the term "bivalent” denote the presence of two binding sites in an antigen binding protein.
  • the term "bivalent, bispecific” as used herein denotes an antigen binding protein that has two antigen-binding sites of which each binds to another antigen (or another epitope of the antigen).
  • Multispecific antigen binding proteins of the present invention have at least two binding sites and are at least bispecific bivalent. In one embodiment of the invention the multispecific antigen binding proteins of the present invention have two binding sites and are bispecific, bivalent.
  • a “scFv fragment” or “single chain Fv fragment” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), and a single-chain-Fv-linker, wherein said antibody domains and said single-chain-Fv-linker have one of the following orders in N- terminal to C-terminal direction: a) VH-single-chain-Fv-linker-VL, b) VL-single- chain-Fv-linker-VH; preferably a) VH-single-chain-Fv-linker-VL, and wherein said single-chain-Fv-linker is a polypeptide of with an amino acid sequence with a length of at least 15 amino acids, in one embodiment with a length of at least 20 amino acids.
  • N-terminus denotes the last amino acid of the N-terminus
  • C-terminus denotes the last amino acid of the C-terminus.
  • single-chain-Fv-linker as used within single chain Fv fragment denotes a peptide with amino acid sequences, which is preferably of synthetic origin. Said single-chain-Fv-linker is a peptide with an amino acid sequence with a length of at least 15 amino acids, in one embodiment with a length of at least 20 amino acids and preferably with a length between 15 and 30 amino acids.
  • said ingle-chain-Fv-linker is (G4S)3 or (G4S)4.
  • single chain Fv fragments are preferably disulfide stabilized.
  • Such further disulfide stabilization of single chain antibodies is achieved by the introduction of a disulfide bond between the variable domains of the single chain antibodies and is described e.g. in WO 94/029350, Rajagopal, V., et al, Protein Eng. 10 (1997) 1453-1459; Kobayashi, H., et al, Nuclear Medicine & Biology 25 (1998) 387-393; or Schmidt, M., et al, Oncogene 18 (1999) 1711 -1721.
  • the disulfide bond between the variable domains of the single chain Fv fragments comprised in the antibody according to the invention is independently for each single chain Fv fragment selected from: i) heavy chain variable domain position 44 to light chain variable domain position 100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100.
  • the disulfide bond between the variable domains of the single chain Fv fragments comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100.
  • a “scFab fragment” or “single chain Fab fragment” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-lmker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
  • Said single chain Fab fragments a) VH-CH1 -linker- VL-CL, b) VL-CL-lmker-VH-CHl, c) VH-CL- linker-VL-CHl and d) VL-CH1 -linker- VH-CL, are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
  • the term "N- terminus” denotes the last amino acid of the N-terminus
  • C-terminus denotes the last amino acid of the C-terminus.
  • linker denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptides according to invention are used to link a) VH-CH1 to VL-CL, b) VL-CL to VH-CH1, c) VH-CL to VL-CH1 or d) VL-CH1 to VH-CL to form the following single chain Fab fragments according to the invention a) VH-CH1 -linker- VL-CL, b) VL-CL-linker- VH-CH1, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH-CL.
  • Said linker within the single chain Fab fragments is a peptide with an amino acid sequence with a length of at least 30 amino acids, preferably with a length of 32 to 50 amino acids.
  • said linker is (G4S)6G2.
  • said antibody domains and said linker in said single chain Fab fragment have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, or b) VL-CL-linker-VH-CHl, more preferably VL-CL- linker-VH-CHl .
  • said antibody domains and said linker in said single chain Fab fragment have one of the following orders in N-terminal to C- terminal direction: a) VH-CL-linker-VL-CHl or b) VL-CHl-linker-VH-CL.
  • the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) are disulfide stabilized by introduction of a disulfide bond between the following positions: i) heavy chain variable domain position 44 to light chain variable domain position
  • the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is between heavy chain variable domain position 105 and light chain variable domain position 43 (numbering always according to EU index of Kabat). In an embodiment single chain Fab fragment without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragments are preferred.
  • the full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes.
  • Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE class (or isotypes) and, in the case of IgG and IgA, their subclasses (or subtypes).
  • an full length antibody of the invention and thus a monovalent antigen binding protein of the invention has a constant domain structure of an IgG class antibody.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition.
  • chimeric antibody refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies”.
  • Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g.,
  • humanized antibody refers to antibodies in which the framework or "complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
  • CDR complementarity determining regions
  • a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody.” See, e.g., Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268-270.
  • Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies.
  • Other forms of "humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences.
  • Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).
  • Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production.
  • Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol.
  • human antibody as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
  • recombinant human antibodies have variable and constant regions in a rearranged form.
  • the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • variable domain (variable domain of a light chain (VL), variable region of a heavy chain (VH) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
  • the domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions” (or complementarity determining regions, CDRs).
  • the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
  • the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • the antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
  • hypervariable region or "antigen-binding portion of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from the "complementarity determining regions” or "CDRs".
  • “Framework” or "FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding.
  • CDR and FR regions are determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • binding refers to the binding of the multispecific antigen binding protein to an epitope of the respective antigen in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE- Healthcare Uppsala, Sweden) with purified wild-type antigen.
  • the affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), k D (dissociation constant), and K D (k D /ka).
  • Binding or specifically binding means a binding affinity (K D ) of 10 ⁇ 8 mo 1/1 or less, preferably 10 ⁇ 9 M to 10 ⁇ 13 mo 1/1.
  • a multispecific antigen binding protein to the invention is specifically binding to each antigen for which it is specific with a binding affinity (K D ) of 10 "8 mol/1 or less, preferably 10 "9 M to 10 "13 mol/1.
  • epitope includes any polypeptide determinant capable of specific binding to a monovalent antigen binding proteins.
  • epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by a monovalent antigen binding protein.
  • an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgGl subclass, or of human IgGl subclass with the mutations L234A and L235A.
  • the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG2 subclass. In a further embodiment the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG3 subclass.
  • the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG4 subclass or, of human IgG4 subclass with the additional mutations S228P and L235E (also named
  • constant region denotes the sum of the domains of an antibody other than the variable region.
  • the constant region is not involved directly in binding of an antigen, but exhibit various effector functions.
  • antibodies are divided in the classes (also named isotypes): IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (also named isotypes), such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2.
  • the heavy chain constant regions that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the light chain constant regions (CL) which can be found in all five antibody classes are called ⁇ (kappa) and ⁇ (lambda).
  • constant region derived from human origin denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region.
  • constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al, Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788).
  • Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which, if altered, provide also reduced Fc receptor binding (Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al, FASEB J. 9 (1995) 115-119; Morgan, A., et al, Immunology 86 (1995) 319-324; EP 0 307 434).
  • an antibody according to the invention has a reduced FcR binding compared to an IgGl antibody and the full length parent antibody is in regard to FcR binding of IgG4 subclass or of IgGl or IgG2 subclass with a mutation in S228, L234, L235 and/or D265, and/ or contains the PVA236 mutation.
  • the mutations in the full length parent antibody are S228P, L234A, L235A, L235E and/or PVA236.
  • the mutations in the full length parent antibody are in IgG4 S228P and L235E and in IgGl L234A and L235A.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • binding of Clq to an antibody is caused by defined protein-protein interactions at the so called binding site.
  • constant region binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al, J. Immunol. 127 (1981) 2555-2560; Bunkhouse, R. and Cobra, J.J., Mol. Immunol.
  • Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
  • ADCC antibody-dependent cellular cytotoxicity
  • complement-dependent cytotoxicity denotes a process initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. Binding of Clq to an antibody is caused by defined protein-protein interactions at the so called binding site.
  • Fc part binding sites are known in the state of the art (see above). Such Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
  • Antibodies of subclass IgGl, IgG2, and IgG3 usually show complement activation including Clq and C3 binding, whereas IgG4 does not activate the complement system and does not bind Clq and/or C3.
  • Cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and US 6,602,684.
  • IgGl type antibodies the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain.
  • ADCC antibody dependent cellular cytotoxicity
  • the monovalent antigen binding protein under A) is characterized in that the modified heavy chains of a) and b) are of IgGl isotype, and the antigen binding protein is afucosylated with an the amount of fucose of
  • the antigen binding protein is afucosylated with an the amount of fucose of 65 % to 5 % of the total amount of oligosaccharides (sugars) at Asn297.
  • afucosylated antigen binding protein refers to an antigen binding protein under A) of IgGl or IgG3 isotype (preferably of IgGl isotype) with an altered pattern of glycosylation in the Fc region at Asn297 having a reduced level of fucose residues.
  • Glycosylation of human IgGl or IgG3 occurs at Asn297 as core fucosylated bianntennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues.
  • These structures are designated as GO, Gl (al,6 or al,3) or G2 glycan residues, depending from the amount of terminal Gal residues (Raju, T.S., BioProcess Int. 1 (2003) 44-53).
  • CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F.H., Glycoconjugate J. 14 (1997) 201-207.
  • Antibodies which are recombinantely expressed in non glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85 %. It should be understood that the term an afucosylated antibody or antigen binding protein as used herein includes an antibody or antigen binding protein having no fucose in its glycosylation pattern. It is commonly known that typical glycosylated residue position in an antibody is the asparagine at position 297 according to the EU numbering system ("Asn297").
  • an afucosylated antigen binding protein under A means an antibody of IgGl or IgG3 isotype (preferably of IgGl isotype) wherein the amount of fucose is 80 % or less (e.g. of 80 % to 1 %) of the total amount of oligosaccharides (sugars) at Asn297 (which means that at least 20 % or more of the oligosaccharides of the Fc region at Asn297 are afucosylated). In one embodiment the amount of fucose is
  • amount of fucose means the amount of said oligosaccharide (fucose) within the oligosaccharide (sugar) chain at Asn297, related to the sum of all oligosaccharides (sugars) attached to Asn 297 (e.g.
  • the oligosaccharides of the Fc region are bisected.
  • the afucosylated antibody or antigen binding protein according to the invention can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity in an amount sufficient to partially fucosylate the oligosaccharides in the Fc region.
  • the polypeptide having GnTIII activity is a fusion polypeptide.
  • al,6-fucosyltransferase activity of the host cell can be decreased or eliminated according to US 6,946,292 to generate glycomodified host cells.
  • the amount of antibody fucosylation can be predetermined e.g. either by fermentation conditions (e.g. fermentation time) or by combination of at least two antibodies with different fucosylation amount.
  • Such afucosylated antigen binding proteins and respective glycoengineering methods are described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P., et al, Nature Biotechnol.
  • glycoengineered antigen binding proteins according to the invention have an increased ADCC (compared to the parent antigen binding proteins).
  • Other glycoengineering methods yielding afucosylated antigen binding proteins according to the invention are described e.g. in Niwa, R.. et al, J. Immunol. Methods 306 (2005) 151-160; Shinkawa, T., et al, J. Biol. Chem, 278 (2003) 3466-3473; WO 03/055993 or US 2005/0249722.
  • EU index (according to Kabat) is generally used when referring to a residue or position in an immunoglobulin heavy chain constant region (e.g., the EU index is reported in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) expressly incorporated herein by reference).
  • the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell
  • NGNA as used within this application denotes the sugar residue
  • N-glycolylneuraminic acid N-glycolylneuraminic acid.
  • the antibody according to the invention is produced by recombinant means.
  • one aspect of the current invention is a nucleic acid encoding the antibody according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention.
  • Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods.
  • the multispecific antigen binding proteins according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA and RNA encoding the multispecific antigen binding proteins is readily isolated and sequenced using conventional procedures.
  • the hybridoma cells can serve as a source of such DNA and RNA.
  • the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant multispecific antigen binding proteins in the host cells.
  • Amino acid sequence variants (or mutants) of the multispecific antigen binding proteins are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
  • host cell denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention.
  • HEK293 cells and CHO cells are used as host cells.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • NS0 cells Expression in NS0 cells is described by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270.
  • Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87.
  • a preferred transient expression system (HEK 293) is described by
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
  • a nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Multispecific antigen binding proteins is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins (e.g. byproducts) by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art (see Ausubel, F., et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).
  • Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g.
  • cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed- mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g.
  • One aspect of the invention is a pharmaceutical composition comprising an multispecific antigen binding protein according to the invention.
  • Another aspect of the invention is the use of a multispecific antigen binding protein according to the invention for the manufacture of a pharmaceutical composition.
  • a further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising a multispecific antigen binding proteins according to the invention.
  • the present invention provides a composition, e.g. a pharmaceutical composition, containing a multispecific antigen binding proteins according to the present invention, formulated together with a pharmaceutical carrier.
  • One embodiment of the invention is the multispecific antigen binding protein according to the invention for the treatment of cancer.
  • Another aspect of the invention is said pharmaceutical composition for the treatment of cancer.
  • Another aspect of the invention is the use of a multispecific antigen binding proteins according to the invention for the manufacture of a medicament for the treatment of cancer.
  • composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • an appropriate carrier for example, liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • cancer refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ure
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier preferably is an isotonic buffered saline solution. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • transformants and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • transfection refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without daunting cell wall barriers are used as host cells, transfection is carried out e.g. by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 546. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride as described by Cohen, F.N, et al, PNAS. 69 (1972) 7110.
  • expression refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins.
  • the transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.
  • a "vector” is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.
  • An "expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide.
  • An "expression system” usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
  • a multispecific antigen binding protein comprising
  • A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
  • the multispecific antigen binding protein according to embodiment 1 characterized in being a bispecific, bivalent antibody.
  • the multispecific antigen binding protein according to any one of claims 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody.
  • the multispecific antigen binding protein according to any one of embodiments 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
  • the multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a Fv fragment, Fab fragment, a scFv fragment, and a scFab fragment.
  • the multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a Fv fragment and Fab fragment which are fused via two peptide connectors to the C-terminus or N-terminus of both heavy chains under A).
  • the multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a scFv fragment and scFab fragment which are fused via one peptide connector to the C-terminus or N-terminus of both heavy chains under A).
  • the multispecific antigen binding protein according to any one of embodiments 5 or 6 characterized in that the antigen binding peptide is a Fv fragment.
  • the multispecific antigen binding protein according to any one of embodiments 1 to 11 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the N-terminus of one or both heavy chains under A).
  • the multispecific antigen binding protein according to any one of embodiments 1 to 11 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus of one or both heavy chains under A).
  • the multispecific antigen binding protein according to embodiment 1 characterized in comprising a) the amino acid sequence of SEQ ID NO: 8;
  • the multispecific antigen binding protein to any one of embodiments 1 to 13 characterized in that the heavy chains under A) are of human IgG isotype.
  • a method for the preparation of a multispecific antigen binding protein according to the invention comprising the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding a multispecific antigen binding protein according to embodiments 1 to 16 b) culturing the host cell under conditions that allow synthesis of said multispecific antigen binding protein molecule; and c) recovering said multispecific antigen binding protein molecule from said culture. 18.
  • Vectors comprising nucleic acid according to embodiment 18 encoding the multispecific antigen binding protein embodiments 1 to 16.
  • a host cell comprising vectors according to embodiment 19.
  • a composition preferably a pharmaceutical or a diagnostic composition of a multispecific antigen binding protein embodiments 1 to 16.
  • a pharmaceutical composition comprising a multispecific antigen binding protein according to embodiments 1 to 16 and at least one pharmaceutically acceptable excipient.
  • DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
  • Desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis.
  • the gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids.
  • the plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing.
  • DNA sequences coding "knobs-into-hole” heavy chain carrying S354C and T366W mutations in the CH3 domain with a C-terminal or N-terminal antigen binding peptide linked by peptide connector(s) as well as "knobs-into-hole” heavy chain carrying Y349C, T366S, L368A and Y407V mutations with a C-terminal or N- terminal antigen binding peptide linked by peptide connector(s) were prepared by gene synthesis with flanking BamHI and Xbal restriction sites. All constructs were designed with a 5 '-end DNA sequence coding for a leader peptide (MGWSCIILFLVATATGVHS), which targets proteins for secretion in eukaryotic cells. Construction of the expression plasmids
  • the following expression vector was used for the construction of all heavy and light chain fusion protein encoding expression plasmids.
  • the vector is composed of the following elements: a hygromycin resistance gene as a selection marker,
  • oriP an origin of replication, oriP, of Epstein-Barr virus (EBV)
  • beta-lactamase gene which confers ampicillin resistance in E. coli, the immediate early enhancer and promoter from the human cytomegalovirus (HCMV),
  • poly A human 1 -immunoglobulin polyadenylation
  • the immunoglobulin fusion genes comprising the heavy chain constucts as well as "knobs-into-hole" constructs with a C-terminal or N-terminal antigen binding peptide linked by peptide connector(s) were prepared by gene synthesis and cloned into pGA18 (ampR) plasmids as described.
  • the pG18 (ampR) plasmids carrying the synthesized DNA segments and the Roche expression vector were digested with BamHI and Xbal restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis. Purified heavy and light chain coding DNA segments were then ligated to the isolated Roche expression vector BamHI/Xbal fragment resulting in the final expression vectors.
  • the final expression vectors were transformed into E. coli cells, expression plasmid DNA was isolated (Miniprep) and subjected to restriction enzyme analysis and DNA sequencing. Correct clones were grown in 150 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep) and sequence integrity confirmed by DNA sequencing.
  • Antibodies were generated by transient transfection of the two plasmids encoding the heavy or modified heavy chain one, respectively and the corresponding heavy chain two using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serumfree FreeStyle 293 expression medium (Invitrogen) were transfected with a mix of the two respective expression plasmids and 293fectin or fectin (Invitrogen). For e.g.
  • HEK293-F cells were seeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8 % C02. The day after the cells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 ⁇ g total plasmid DNA (1 ⁇ g/mL) encoding the heavy or modified heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM + 1.2 mL 293 fectin or fectin (2 ⁇ /mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.
  • the protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et. al, Protein Science, 1995, 4, 2411-1423.
  • the concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with Protein A Agarose-beads (Roche). 60
  • Protein A Agarose beads are washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1 - 15 mL cell culture supernatant are applied to the Protein A Agarose beads pre-equilibrated in TBS- NP40. After incubation for at 1 h at room temperature the beads are washed on an Ultrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2x phosphate buffered saline (2xPBS, Roche) and briefly four times with 0.5 mL 100 mM Na-citrate pH 5,0.
  • TBS-NP40 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40.
  • 1 - 15 mL cell culture supernatant are applied to the Protein A Agarose beads pre-equilibrated in TBS-
  • Bound antibody is eluted by addition of 35 ⁇ NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample is combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated for 10 min at 70°C. Consequently, 20 ⁇ are applied to an 4-12 % NuPAGE® Bis-Tris
  • SDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.
  • the concentration of antibodies and derivatives in cell culture supernatants was measured by Protein A-HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to Protein A were applied to a HiTrap Protein A column (GE Healthcare) in 50 mM K2HP04, 300 mM NaCl, pH 7.3 and eluted from the matrix with 550 mM acetic acid, pH 2.5 on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. A purified standard IgGl antibody served as a standard.
  • the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche) were coated with 100 ⁇ biotinylated anti-human IgG capture molecule F(ab')2 ⁇ h-Fcgamma> BI (Dianova) at 0.1 ⁇ g/mL for 1 h at room temperature or alternatively over night at 4°C and subsequently washed three times with 200 ⁇ PBS, 0.05 % Tween (PBST, Sigma).
  • PBST 0.05 % Tween
  • Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS. Elution of antibodies was achieved at acidic pH followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM Histidine, 140 mM NaCl pH 6.0. Monomeric antibody fractions were pooled, concentrated if required using e.g. a
  • the NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 4 - 20 % NuPAGE® Novex® TRIS- Glycine Pre-Cast gels and a Novex® TRIS-Glycine SDS running buffer were used. Reducing of samples was achieved by adding NuPAGE® sample reducing agent prior to running the gel.
  • SDS-PAGE sodium dodecylsulfate-based polyacrylamide gel electrophoresis
  • CE-SDS allows quantification of the individual components.
  • Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2P04/K2HP04, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2 x PBS on a Dionex HPLC-System.
  • the eluted protein was quantified by UV absorbance and integration of peak areas.
  • BioRad Gel Filtration Standard 151 - 1901 served as a standard, (see e.g. Figure 4).
  • SEC-MALLS size-exclusion chromatography with multi-angle laser light scattering
  • concentration-sensitive detectors can be e.g. UV absorption detectors or differential refractive index (RI) detectors.
  • SEC-MALLS typically gives rise to molecular weight estimates with an accuracy that allows clear discrimination between monomers, dimers, trimers etc., provided the SEC separation is sufficient.
  • the total deglycosylated mass of antibodies was determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 ⁇ g purified antibodies were deglycosylated with 50 mU N-Glycosidase F (PNGaseF,
  • ProZyme in 100 mM KH2P04/K2HP04, pH 7 at 37°C for 12-24 h at a protein concentration of up to 2 mg/ml and subsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare).
  • the mass of the respective heavy and light chains was determined by ESI-MS after deglycosylation and reduction.
  • 50 ⁇ g antibody in 115 ⁇ were incubated with 60 ⁇ 1M TCEP and 50 ⁇ 8 M Guanidine- hydrochloride subsequently desalted.
  • the total mass and the mass of the reduced heavy and light chains was determined via ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source.
  • VEGF165-His protein The binding properties of the antibodies was evaluated in an ELISA assay with full-length VEGF165-His protein (R&D Systems) ( Figure 5).
  • Falcon polystyrene clear enhanced microtiter plates were coated with 100 ⁇ 2 ⁇ g/mL recombinant human VEGF 165 (R&D Systems) in PBS for 2 h at room temperature or over night at 4°C.
  • the wells were washed three times with 300 ⁇ PBST (0,2% Tween 20) and blocked with 200 ⁇ 2 % BSA 0,1 % Tween 20 for 30 min at room temperature and subsequently washed three times with 300 ⁇ 1 PBST.
  • Unbound detection antibody was washed away three times with 300 ⁇ PBST and the bound detection antibody was detected by addition of 100 ⁇ , ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
  • Unbound detection antibody was washed away three times with 300 ⁇ , ⁇ PBST and the bound detection antibody was detected by addition of 100 ⁇ , ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
  • the binding properties of antibodies were evaluated in an ELISA assay with full- length Angiopoietin-2-His protein (R&D Systems #623-AN/CF or in house produced material) or Angiopoietin-l-His (R&D systems #923 -AN). Therefore 96 well plates (Falcon polystyrene clear enhanced microtiter plates or Nunc Maxisorb) were coated with 100 ⁇ 1 ⁇ g/mL recombinant human Angiopoietin-1 or
  • Angiopoietin-2 (carrier-free) in PBS (Sigma) for 2 h at room temperature or over night at 4°C.
  • the wells were washed three times with 300 ⁇ 1 PBST (0,2 % Tween 20) and blocked with 200 ⁇ 2 % BSA 0,1 % Tween 20 for 30 min at room temperature and subsequently washed three times with 300 ⁇ 1 PBST.
  • 100 ⁇ , ⁇ of a dilution series (40 pM-0.01 pM) of purified test antibody in PBS was added to the wells and incubated for 1 h on a microtiterplate shaker at room temperature.
  • the interaction ELISA was performed on 384 well microtiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After each incubation step plates were washed 3 times with PBST. ELISA plates were coated with 0.5 ⁇ g/ml Tie-2 protein (R&D Systems, UK, Cat.No.313-TI) for at least 2 hours (h). Thereafter the wells were blocked with PBS supplemented with 0.2 % Tween-20 and 2 % BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions of purified antibodies in PBS were incubated together with 0.2 ⁇ g/ml huAngiopoietin-2 (R&D Systems, UK, Cat.No.
  • Binding of the antibodies to the antigen e.g. human ANG-2 were investigated by surface plasmon resonance using a BIACORE T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements goat ⁇ hIgG- Fcy> polyclonal antibodies were immobilized on a CM5 chip via amine coupling for presentation of the antibodies against human ANG-2 ( Figure 6B). Binding was measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005 % Tween 20, ph 7.4), 25°C.
  • HBS buffer HBS-P (10 mM HEPES, 150 mM NaCl, 0.005 % Tween 20, ph 7.4
  • ANG-2-His (R&D systems or in house purified) was added in various concentrations between 6,25 nM and 200 nM in solution. Association was measured by an ANG-2-injection of 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3 minutes and a KD value was estimated using a 1 : 1 Langmuir binding model. Due to heterogenity of the ANG-2 preparation no 1 : 1 binding could be observed; KD values are thus only relative estimations. Negative control data (e.g. buffer curves) were subtracted from sample curves for correction of system intrinsic baseline drift and for noise signal reduction. Biacore T100 Evaluation Software version 1.1.1 was used for analysis of sensorgrams and for calculation of affinity data.
  • Ang-2 could be captured with a capture level of 2000-1700 RU via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen No. 34660) that was immobilized on a CM5 chip via amine coupling (BSA-free) (see below).
  • PentaHisAntibody PentaHis-Ab BSA-free, Qiagen No. 34660
  • BSA-free amine coupling
  • VEGF binding Kinetic characterization of VEGF binding at 37°C by surface plasmon resonance (Biacore)
  • VEGF-ANG2> antibodies were captured on a CM5-Chip via binding to a Goat Anti Human IgG (JIR 109-005-098).
  • the capture antibody was immobilized by amino coupling using standard amino coupling as follows: HBS-N buffer served as running buffer, activation was done by mixture of
  • EDC/NHS with the aim for a ligand density of 700 RU.
  • rhVEGF (rhVEGF, R&D-Systems Cat.- No, 293-VE) was used as analyte.
  • the kinetic characterization of VEGF binding to ⁇ VEGF-ANG2> antibodies was performed at 37°C in PBS + 0.005 % (v/v) Tween20 as running buffer.
  • the sample was injected with a flow of 50 ⁇ / ⁇ and an association of time 80 sec. and a dissociation time of 1200 sec with a concentration series of rhVEGF from 300 - 0.29 nM.
  • Regeneration of free capture antibody surface was performed with 10 mM Glycin pH 1.5 and a contact time of 60 sec after each analyte cycle.
  • Kinetic constants were calculated by using the usual double referencing method (control reference: binding of rhVEGF to capture molecule Goat Anti Human IgG, blanks on the measuring flow cell, rhVEGF concentration "0", Model: Langmuir binding 1 : 1, (Rmax set to local because of capture molecule binding).
  • Figure 11 shows a schematic view of the Biacore assay.
  • HEK293-Tie cell line In order to determine the interference of Angiopoietin-2 antibodies with ANGPT2 (ANG2) stimulated Tie2 phosphorylation and binding of ANGPT2 to Tie2 on cells a recombinant HEK293-Tie cell line was generated. Briefly, a pcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2) coding for full-length human Tie2 (SEQ ID 108) under control of a CMV promoter and a Neomycin resistance marker was transfected using Fugene (Roche Applied Science) as transfection reagent into HEK293 cells (ATCC) and resistant cells were selected in DMEM 10 % FCS, 500 ⁇ g/ml G418.
  • Fugene Roche Applied Science
  • ANGPT2 ANG2 induced Tie2 phosphorylation by ⁇ VEGF-ANG2> antibodies was measured according to the following assay principle.
  • HEK293-Tie2 clone22 was stimulated with ANGPT2 for 5 minutes in the absence or presence of ANGPT2 antibody and P-Tie2 was quantified by a sandwich ELISA.
  • 2x105 HEK293-Tie2 clone 22 cells per well were grown over night on a Poly-D- Lysine coated 96 well- microtiter plate in ⁇ DMEM, 10% FCS, 500 ⁇ Geneticin.
  • the next day a titration row of ANGPT2 antibodies was prepared in a microtiter plate (4-fold concentrated, 75 ⁇ final volume/well, duplicates) and mixed with 75 ⁇ 1 of an ANGPT2 (R&D systems # 623-AN] dilution (3.2 ⁇ as 4-fold concentrated solution). Antibodies and ANGPT2 were pre-incubated for 15 min at room temperature.
  • Tris pH 8.0, 137 mM NaCl, 1 % NP-40, 10 % glycerol, 2mM EDTA, I mM NaV304, 1 mM PMSF and 10 ⁇ Aprotinin) per well on ice.
  • Cells were lysed for 30 min at 4°C on a microtiter plate shaker and 100 ⁇ lysate were transferred directly into a p-Tie2 ELISA microtiter plate (R&D Systems, R&D #DY990) without previous centrifugation and without total protein determination.
  • P-Tie2 amounts were quantified according to the manufacturer's instructions and IC50 values for inhibition were determined using XLfit4 analysis plug-in for Excel (Dose-response one site, model 205). IC50 values can be compared within on experiment but might vary from experiment to experiment.
  • VEGF induced HUVEC proliferation assay
  • VEGF induced HUVEC Human Umbilical Vein Endothelial Cells, Promocell #C- 12200 proliferation was chosen to measure the cellular function of VEGF antibodies. Briefly, 5000 HUVEC cells (low passage number, ⁇ 5 passages) per 96 well were incubated in ⁇ starvation medium (EBM-2 Endothelial basal medium 2, Promocell # C-22211, 0.5% FCS, Penicilline/Streptomycine) in a collagen I-coated BD Biocoat Collagen I 96-well microtiter plate (BD #354407 / 35640 over night.
  • ⁇ starvation medium EBM-2 Endothelial basal medium 2, Promocell # C-22211, 0.5% FCS, Penicilline/Streptomycine
  • mice 8 to 10 weeks old female Balb/c mice were purchased from Charles River, Sulzfeld, Germany.
  • the protocol was modified according to the method described by Rogers et al. (2007). Briefly, micropockets with a width of about 500 ⁇ were prepared under a microscope at approximately 1 mm from the limbus to the top of the cornea using a surgical blade and sharp tweezers in the anesthetized mouse.
  • the disc (Nylaflo®, Pall Corporation, Michigan) with a diameter of 0.6 mm was implanted and the surface of the implantation area was smoothened. Discs were incubated in corresponding growth factor or in vehicle for at least 30 min. After 3, 5 and 7 days, eyes were photographed and vascular response was measured. The assay was quantified by calculating the percentage of the area of new vessels per total area of the cornea.
  • the bispecific, bivalent antigen binding proteins according to the invention binding to VEGF (VEGF-A) and ANG-2 (Angiopoietin-2) comprise a first antigen-binding site that binds to VEGF and a second antigen-binding site that binds to ANG-2.
  • first antigen-binding site binding to VEGF e.g. the heavy chain variable domain of the light chain variable domains from the anti-VEGF antibodies ⁇ VEGF>bevacizumab (see e.g. WO2010/040508 which incorporated by reference).
  • As second antigen-binding site comprises the heavy chain variable domains and the light chain variable domains from the anti-ANG-2 antibodies ⁇ ANG-2> Ang2s_R3_LC03, ⁇ ANG-2>Ang2i_LC06, ⁇ ANG-2>Ang2i_LC07, ⁇ ANG-2> Ang2k_LC08, ⁇ ANG-2> Ang2s_LC09, ⁇ ANG-2> Ang2i_LC10, or ⁇ ANG-2> Ang2k_LCl l, preferably from ⁇ ANG-2>Ang2i_LC06, or ⁇ ANG-2> Ang2k_LC08 bevacizumab ( see e.g. WO 2010/040508 which incorporated by reference).
  • novel bispecific monovalent antibody-derived protein entities were constructed.
  • the basis for this novel bispecific, bivalent antigen binding proteins are monovalent antibodies (MoAb) as described e.g. in US 2004/0033561 and PCT Application PCT/EP2012/053119. They consist of 2 different heavy chains: i) a) a first heavy chain of an full length antibody; and
  • heavy chain 1 is a typical IgG like heavy chain consisting of VH-CH1- CH2-CH3 domains.
  • Heavy chain 2 (HC2) consists of VL-CL-CH2-CH3 domains.
  • heavy chain 1 (HC1) is a typical IgG like heavy chain consisting of VH-CL- CH2-CH3 domains.
  • Heavy chain 2 (HC2) consists of VL-CH 1 -CH2-CH3 domains
  • BiMoMAb bispecific, bivalent antigen binding protein according to the invention
  • the heavy chain Fab domain (VH-CH1) and the light chain Fab domain (VL-CL) of the respective antibody were linked by a glycine serine (G4S)3 or (G4S)4 single-chain-linker to give a single chain Fv (scFab), which was attached to the C- terminus of the other antibody heavy chain using e.g. a (G4S)3-connector.
  • G4S glycine serine
  • scFab single chain Fv
  • cysteine residues were introduced in the VH (including Kabat position 44,) and VL (including Kabat position 100) domain of the scFv binding to ANG-2 or VEGF as described earlier (e.g. WO 94/029350; Reiter et al, Nature Biotechnology (1996) 1239-1245; Young et al, FEBS Letters (1995) 135-139; or Rajagopal et al, Protein Eng. (1997) 1453-59).
  • VEGF MoAb VEGF monovalent antibody
  • hole VEGF MoAb heavy chain 1
  • knock VEGF MoAb heavy chain 2
  • the bispecific, bivalent antigen binding protein of the invention BiMoMAb-N-Fab is made by fusing the Ang2i-LC06-CL-VL (SEQ ID NO: 4) via a (G4S)4 connector to the VEGF MoAb heavy chain 1 (hole) (SEQ ID NO: l) and the Ang2i-LC06-CHl-VH (SEQ ID NO: 3) domain via a (G4S)4 connector to the VEGF MoAb heavy chain 2 (knob) (SEQ ID NO: 2).
  • the bispecific, bivalent antigen binding protein of the invention BiMoMAb-N-Fv is made by fusing the Ang2i-Vl (SEQ ID NO: 6) via a (G4S)4 connector to the N-terminus of HC1 (SEQ ID NO: l)of the VEGF MoAb and the Ang2i-LC06-Vh (SEQ ID NO: 5) domain via a (G4S)4 connector to the HC2 (SEQ ID NO: 2)of the VEGF MoAb.
  • the bispecific, bivalent antigen binding protein of the invention BiMoMAb-C-scFab is made by fusing the Ang2i-LC06 scFab (SEQ ID NO: 7) via a (G4S)4 connector to the C-terminus of HC2 (SEQ ID NO: 2) of the VEGF MoAb and the VEGF-MoAb HC1 (SEQ ID NO: 1 remains unchanged (the SEQ ID NO: 1 is identical to SEQ ID NO: 12, see Sequence listing).
  • Table la Different bispecific, bivalent antigen binding proteins of the invention comrising as first antigen binding site under A) i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; and as second antigen binding site under B) different antigen binding peptides
  • Heavy chain 1 and heavy chain 2 of the corresponding bispecific monovalent antibodies BiMoMAb 1, BiMoMAb 2 and BiMoMAb 3 and BiMOMab 4 were constructed in genomic expression vectors as described above).
  • the plasmids were amplified in E. coli, purified, and subsequently transfected for transient expression of recombinant proteins in HEK293-F cells (utilizing Invitrogen's FreeStyle 293 system). After 7 days, HEK 293-F cell supernatants were harvested, filtered and the bispecific antibodies were purified by protein A and size exclusion chromatography. Homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non reducing and reducing conditions and analytical size exclusion chromatography. The expression yields were summarized in Table 2.
  • Table 2 summarizes the purifications of different BiMoMAb constructs.
  • the preparative SEC shows two prominent HMW peaks and one monomer peak for the BiMoMAb-N-Fab.
  • the BiMoMAb-C-scFab shows two small HMW peaks and one well defined monomer peak.
  • Chip based CE-SDS analysis shows for the BiMoMAb-N-Fab one band under reducing conditions with an apparent molecular weight of 97 kDa and 160 kDa under non reducing conditions.
  • SDS-PAGE analysis of the BiMoMAb-C-scFab under reducing conditions shows two polypeptide heavy chains one carrying the C-terminal scFab fusion with a apparent molecular weight of 100 kDa and the other heavy chain without a fusion partner with an apparent molecular weight of 50 kDa.
  • bispecific antibody as analyte in solution In this assay the bispecific
  • the association rate for the BiMoMAb-N-Fab seems to be slower than the association rate for the BiMoMAb-C-scFab (4E4 versus 1E5 M ⁇ V l) and for the the VEGF binding affinity is about that is described above that it binds to VEGF comparable to its parent antibody.
  • Table 5 Biacore binding of different multispecific antigen binding proteins of the invention to VEGF
  • BimoMab-C-scFab 1.01E+05 ⁇ 1E-06 ⁇ lE-10 150.00
  • bispecific antibodies BiMoMAb-N-Fab and BiMoMAb-C- scFab showed a dose-dependent interference with ANGPT2 stimulated Tie2 phosphorylation.
  • the IC50 values were higher than the ones obtained for bivalent binding molecules like the originating IgG (Ang2i-LC06) but comparable to those of the Fabs from Ang2i-LC06.
  • the BiMoMAb-N-Fab interfered with ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx. 3320 ng/ml
  • BiMoMAb-C-scFab interfered with ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx. 1504 ng/ml.
  • the IC50 values are higher for monovalent binders, then for bivalent binder, but in a typical range for monovalent (Fab) binding and comparable to their mother clones Ang2i-LC06 as a Fab within the error of this cellular assay.
  • the interaction ELISA is performed on 384 well microtiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After each incubation step plates are washed 3 times with PBST. ELISA plates were coated with 0.5 ⁇ g/ml Tie-2 protein (R&D Systems,
  • BAM0981 (R&D Systems, UK) and 1 :3000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat.No.l 1089153001) is added for 1 h. Thereafter the plates are washed 6 times with PBST. Plates are developed with freshly prepared ABTS reagent (Roche Diagnostics GmbH, DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT. Absorbance was measured at 405 nm.

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Abstract

The present invention relates to multispecific antigen binding proteins, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Description

Multispecific antigen binding proteins
The present invention relates to multispecific antibody, especially to a bispecific, bivalent antibody (comprising as one antigen binding site a monovalent antigen binding protein), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof. Background of the Invention
In the last two decades various engineered antibody derivatives , either mono or- multispecific, either mono- or multivalent have been developed and evaluated (see e.g. Holliger, P., et al, Nature Biotech 23 (2005) 1126-1136; Fischer, N., and Leger, O., Pathobiology 74 (2007) 3-14). For certain antigens as e.g. c-Met monovalent antibodies have different properties such as lack of agonistic function or reduced receptor internalization upon antibody binding than their corresponding bivalent forms and therefore represent attractive formats for therapeutic use. E.g. WO 2005/063816 refers to monovalent antibody fragments as therapeutics. US 2004/0033561 describes a method for the generation of monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL- CH2-CH3 antibody chain; however, a disadvantage of this method is the formation of a binding inactive homodimer of VL-CL-CH2-CH3 chains. Due the similar molecular weight such homodimeric by-products are the difficult to separate. WO 2007/048037 also refers to monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain, but having a tagging moiety attached to the heavy chain for easier purification of the heterodimer from the difficult-to-separate homodimeric by-product. WO 2009/089004 describes another possibility to generate a heterodimeric monovalent antibody using electrostatic steering effects.
Summary of the Invention
The invention comprises a multispecific antigen binding protein, comprising
A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
B) an antigen binding peptide which specifically binds to a second antigen, wherein the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus or N-terminus of one or both heavy chains under A).
In one embodiment the multispecific antigen binding protein according to the invention is a bispecific, bivalent antibody.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and
b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a Fv fragment, Fab fragment, a scFv fragment, and a scFab fragment. In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a Fv fragment and Fab fragment which are fused via two peptide connectors to the C-terminus or N-terminus of both heavy chains under A). In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is selected from the group of a scFv fragment and scFab fragment which are fused via one peptide connector to the C-terminus or N-terminus of both heavy chains under
A) . In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a Fv fragment.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a Fab fragment.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a scFv fragment.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide is a scFab fragment.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide under
B) is fused via one or two peptide connectors to the N-terminus of one or both heavy chains under A).
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus of one or both heavy chains under A). In one embodiment the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and b) the amino acid sequence of SEQ ID NO:9;
or a) the amino acid sequence of SEQ ID NO: 10; and
b) the amino acid sequence of SEQ ID NO: 11 ;
or a) the amino acid sequence of SEQ ID NO: 12; and
b) the amino acid sequence of SEQ ID NO: 13;
or a) the amino acid sequence of SEQ ID NO: 14; and
b) the amino acid sequence of SEQ ID NO: 15;.
In one aspect of the invention the multispecific antigen binding protein according to the invention is characterized in that the heavy chains under A) are of human IgG isotype.
In one aspect of the invention the multispecific antigen binding protein according to the invention is characterized in that the heavy chains under A) are of human IgGl or IgG4 isotype.
The invention further comprises a method for the preparation of a multispecific antigen binding protein according to the invention comprising the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding
a monovalent antigen binding protein according to the invention b) culturing the host cell under conditions that allow synthesis of said multispecific antigen binding protein molecule; and
c) recovering said multispecific antigen binding protein molecule from said culture.
The invention further comprises nucleic acid encoding the multispecific antigen binding protein according to the invention. The invention further comprises vectors comprising nucleic acid encoding the multispecific antigen binding protein according to the invention.
The invention further comprises host cell comprising said vectors.
The invention further comprises composition, preferably a pharmaceutical or a diagnostic composition of a multispecific antigen binding protein according to the invention.
The invention further comprises pharmaceutical composition comprising a multispecific antigen binding protein according to the invention and at least one pharmaceutically acceptable excipient. The invention further comprises method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of a multispecific antigen binding protein binding protein according to the invention.
The multispecific antigen binding proteins according to the invention have valuable properties like low aggregation tendency, high stability, valuable pharmacokinetic properties (like e.g. halftime (term tl/2) or AUC) and are producible in good yields.
It has further been found that the multispecific antigen binding proteins according to the invention have valuable characteristics such as biological or pharmacological activities (as e.g. ADCC, or antagonistic biological activity as well as lack of agonistic activities). They can be used e.g. for the treatment of diseases such as cancer.
Description of the Figures
Figure 1 A - H) Scheme of different multispecific antigen binding protein according to the invention comprising a monovalent antigen binding protein (under A) i)) with VL-CL-CH2-CH3 chain and VH-CH1-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen. Figure 2 A - H) Scheme of different multispecific antigen binding protein according to the invention comprising a monovalent antigen binding protein (under A) ii)) with VL-CH1-CH2-CH3 chain and VH-CL-CH2-CH3 chain from a full length antibody specifically binding to a first antigen and an antigen binding peptide (scFv, scFab, Fv or Fab) specifically binding to a second antigen.
Figure 3 Preparative SEC Chromatograms of A) BimoMab-N-Fab and B)
BimoMab-C-scFab.
Detailed Description of the Invention
The invention comprises a multispecific antigen binding protein, comprising
A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and
b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
B) an antigen binding peptide which specifically binds to a second antigen, wherein the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus or N-terminus of one or both heavy chains under A).
In one embodiment the multispecific antigen binding protein according to the invention is a bispecific, bivalent antibody.
In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody. In one embodiment of the invention the multispecific antigen binding protein according to the invention is characterized in that the a monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
In one preferred embodiment of the invention the CH3 domains of said monovalent antigen binding protein under A can be altered by the "knobs-into-holes" (KiH) technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al, Protein Eng. 9 (1996) 617-621; and
Merchant, A.M., et al, Nat. Biotechnol. 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerisation of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A.M., et al, Nat. Biotechnol. 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and increases the yield.
Thus in one aspect of the invention said monovalent antigen binding protein under A), which specifically binds to a first antigen, is further characterized in that the CH3 domain of the heavy chain of the full length antibody of a) and the CH3 domain of the modified heavy chain of the full length antibody of b) each meet at an interface which comprises an original interface between the antibody CH3 domains;
wherein said interface is altered to promote the formation of the monovalent antigen binding protein, wherein the alteration is characterized in that: i) the CH3 domain of one heavy chain is altered,
so that within the original interface the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the monovalent antigen binding protein,
an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and
ii) the CH3 domain of the other heavy chain is altered,
so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the monovalent antigen binding protein,
an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.
Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).
In one aspect of the invention both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.
In one preferred embodiment, said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises a T366W mutation in the CH3 domain of the "knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain". An additional interchain disulfide bridge between the
CH3 domains can also be used (Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and a E356C mutation or a S354C mutation into the CH3 domain of the "hole chain". Thus in a another preferred embodiment, said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C,
T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering always according to EU index of Kabat). But also other knobs-in-holes technologies as described by EP 1 870 459 Al, can be used alternatively or additionally. A preferred example for said monovalent antigen binding protein are R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain" (numbering always according to EU index of Kabat). In general, there different modifications known in the art to enhance the formation of the heterodimer, which can be used as alternatives, these different modifications are as described e.g. in WO 96/027011, Ridgway, J.B., et al, Protein Eng. 9 (1996) 617-621; Merchant, A.M., et al, Nat. Biotechnol. 16 (1998) 677-681, WO 96/027011, WO 98/050431, US 2010/0015133, WO 2007/147901, WO 2009/089004, WO 2010/129304 and Muda, M., et al, Protein Eng., Design & Selection 24 (2011) 447-454.
In another preferred embodiment said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises a T366W mutation in the CH3 domain of the "knobs chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain" and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain".
In another preferred embodiment said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or said monovalent antigen binding protein under A), which specifically binds to a first antigen comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally R409D; K370E mutations in the CH3 domain of the "knobs chain" and D399K; E357K mutations in the CH3 domain of the "hole chain". In one embodiment the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and
b) the amino acid sequence of SEQ ID NO:9. In one embodiment the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 10; and
b) the amino acid sequence of SEQ ID NO: 11.
In one embodiment the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 12; and
b) the amino acid sequence of SEQ ID NO: 13.
In one embodiment the multispecific antigen binding protein according to the invention is characterized in comprising a) the amino acid sequence of SEQ ID NO: 14; and
b) the amino acid sequence of SEQ ID NO: 15.
The term "full length antibody" as used herein denotes a full length antibody consisting of two antibody heavy chains and two antibody light chains (see Fig. 1). A heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH 1 -HR-CH2-CH3 ; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE. Preferably the heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of VH, CHI, HR, CH2 and CH3. The light chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHI domain (i.e. between the light and heavy chain) and between the hinge regions of the full length antibody heavy chains. Examples of typical full length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE.) Such full length antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies. The full length antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same (first) antigen.
From these full length antibodies the monovalent antigen binding protein under A), which specifically binds to a first antigen are derived by modifying: a) the first heavy chain of said antibody by replacing the VH domain by the VL domain of said antibody; and by modifying b) the second heavy chain of said antibody by replacing the CHI domain by the CL domain of said antibody. Thus the monovalent antigen binding protein under A), which specifically binds to a first antigen comprise two modified heavy chains and no light chains. The term "antigen binding peptide" as used refers to a monovalent antigen binding fragment or derivative of a full length antibody which includes an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), or a pair of VH/VL derived from full length antibodies or antibody fragments such as a VH domain and/or a VL domain, a Fv fragment, a single chain Fv (scFv) fragment, a Fab fragment, or single chain Fab (scFab) fragment. In one embodiment the antigen binding peptide is a single variable heavy chain domain (sVH) with sufficient binding affinity to specifically bind to the respective antigen. In one embodiment the antigen binding peptide comprises at least an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In another embodiment such antigen binding peptides are selected from the group consisting of a VH domain, a single chain Fv (scFv) fragment, and a single chain Fab (scFab) fragment, preferably from the group consisting of Fv fragment, a Fab fragment, a single chain Fv (scFv) fragment and a single chain Fab (scFab) fragment. The term "peptide connector" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptide connectors according to invention are used to fuse the antigen binding peptides to the C-or N-terminus of the full length and/or modified full length antibody chains to form a trispecific or tetraspecific antibody according to the invention. Preferably said peptide connectors under c) are peptides with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment said peptide connector is (GxS)n or (GxS)nGm with G = glycine, S = serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In one embodiment said peptide connector is
(G4S)2.
The C-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the C-terminus of said heavy or light chain.
The terms "binding site" or "antigen-binding site" as used herein denotes the region(s) of antigen binding protein according to the invention to which a ligand (e.g the antigen or antigen fragment of it) actually binds and which is derived from antibody molecule or a fragment thereof (e.g. a Fab fragment). The antigen-binding site according to the invention comprise an antibody heavy chain variable domains (VH) and an antibody light chain variable domains (VL). The antigen-binding sites (i.e. the pairs of VH/VL) that specifically bind to the desired antigen can be derived a) from known antibodies to the antigen or b) from new antibodies or antibody fragments obtained by de novo immunization methods using inter alia either the antigen protein or nucleic acid or fragments thereof or by phage display. An antigen-binding site of a monovalent antigen binding protein of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRLl, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.
Antibody or antigen binding protein specificity refers to selective recognition of the antibody or antigen binding protein for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. Multispecific antigen binding proteins have more than one antigen-binding specificities and are e.g. bispecific , tri- or tetraspecific. In one embodiment of the invention they are bispecific, in one embodiment they are bispecific, bivalent. Bispecific antigen binding proteins have two different antigen-binding specificities. Trispecific antigen binding proteins accordingly have three different antigen-binding specificities. Tetraspecific antigen binding proteins have four different antigen-binding specificities. The term "valent" as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule or antigen binding protein. A natural antibody for example or a full length antibody according to the invention has two binding sites and is bivalent. The term monovalent refers to one binding site in an antigen binding protein, therefore to only one specificity. As such, the term "bivalent", denote the presence of two binding sites in an antigen binding protein. The term "bivalent, bispecific" as used herein denotes an antigen binding protein that has two antigen-binding sites of which each binds to another antigen (or another epitope of the antigen). Multispecific antigen binding proteins of the present invention have at least two binding sites and are at least bispecific bivalent. In one embodiment of the invention the multispecific antigen binding proteins of the present invention have two binding sites and are bispecific, bivalent. A "scFv fragment" or "single chain Fv fragment" (see Fig2b) is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), and a single-chain-Fv-linker, wherein said antibody domains and said single-chain-Fv-linker have one of the following orders in N- terminal to C-terminal direction: a) VH-single-chain-Fv-linker-VL, b) VL-single- chain-Fv-linker-VH; preferably a) VH-single-chain-Fv-linker-VL, and wherein said single-chain-Fv-linker is a polypeptide of with an amino acid sequence with a length of at least 15 amino acids, in one embodiment with a length of at least 20 amino acids. The term "N-terminus denotes the last amino acid of the N-terminus, The term "C-terminus denotes the last amino acid of the C-terminus. The term "single-chain-Fv-linker" as used within single chain Fv fragment denotes a peptide with amino acid sequences, which is preferably of synthetic origin. Said single-chain-Fv-linker is a peptide with an amino acid sequence with a length of at least 15 amino acids, in one embodiment with a length of at least 20 amino acids and preferably with a length between 15 and 30 amino acids. In one embodiment said single-chain-linker is (GxS)n with G=glycine, S=serine, (x=3 and n=4, 5 or 6) or (x=4 and n=3, 4, 5 or 6), preferably with x=4, n=3, 4 or 5, more preferably with x=4, n=3 or 4. In one embodiment said ingle-chain-Fv-linker is (G4S)3 or (G4S)4.
Furthermore said single chain Fv fragments are preferably disulfide stabilized. Such further disulfide stabilization of single chain antibodies is achieved by the introduction of a disulfide bond between the variable domains of the single chain antibodies and is described e.g. in WO 94/029350, Rajagopal, V., et al, Protein Eng. 10 (1997) 1453-1459; Kobayashi, H., et al, Nuclear Medicine & Biology 25 (1998) 387-393; or Schmidt, M., et al, Oncogene 18 (1999) 1711 -1721.
In one embodiment of the disulfide stabilized single chain Fv fragments, the disulfide bond between the variable domains of the single chain Fv fragments comprised in the antibody according to the invention is independently for each single chain Fv fragment selected from: i) heavy chain variable domain position 44 to light chain variable domain position 100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100.
In one embodiment the disulfide bond between the variable domains of the single chain Fv fragments comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100.
A "scFab fragment" or "single chain Fab fragment" (see Fig2a ) is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-lmker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments a) VH-CH1 -linker- VL-CL, b) VL-CL-lmker-VH-CHl, c) VH-CL- linker-VL-CHl and d) VL-CH1 -linker- VH-CL, are stabilized via the natural disulfide bond between the CL domain and the CHI domain. The term "N- terminus" denotes the last amino acid of the N-terminus, The term "C-terminus" denotes the last amino acid of the C-terminus.
The term "linker" as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptides according to invention are used to link a) VH-CH1 to VL-CL, b) VL-CL to VH-CH1, c) VH-CL to VL-CH1 or d) VL-CH1 to VH-CL to form the following single chain Fab fragments according to the invention a) VH-CH1 -linker- VL-CL, b) VL-CL-linker- VH-CH1, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH-CL. Said linker within the single chain Fab fragments is a peptide with an amino acid sequence with a length of at least 30 amino acids, preferably with a length of 32 to 50 amino acids. In one embodiment said linker is (GxS)n with G=glycine, S=serine, (x=3, n=8, 9 or 10 and m=0, 1, 2 or 3) or (x=4 and n=6, 7 or 8 and m=0, 1, 2 or 3), preferably with x=4, n=6 or 7 and m=0, 1, 2 or 3, more preferably with x=4, n=7 and m=2. In one embodiment said linker is (G4S)6G2.
In a preferred embodiment said antibody domains and said linker in said single chain Fab fragment have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, or b) VL-CL-linker-VH-CHl, more preferably VL-CL- linker-VH-CHl .
In another preferred embodiment said antibody domains and said linker in said single chain Fab fragment have one of the following orders in N-terminal to C- terminal direction: a) VH-CL-linker-VL-CHl or b) VL-CHl-linker-VH-CL. Optionally in said single chain Fab fragment, additionally to the natural disulfide bond between the CL-domain and the CHI domain, also the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) are disulfide stabilized by introduction of a disulfide bond between the following positions: i) heavy chain variable domain position 44 to light chain variable domain position
100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100 (numbering always according to EU index of Kabat). Such further disulfide stabilization of single chain Fab fragments is achieved by the introduction of a disulfide bond between the variable domains VH and VL of the single chain Fab fragments. Techniques to introduce unnatural disulfide bridges for stabilization for a single chain Fv are described e.g. in WO 94/029350, Rajagopal, V., et al, Protein Eng. 10 (1997) 1453-1459; Kobayashi, H., et al,
Nuclear Medicine & Biology 25 (1998) 387-393; or Schmidt, M., et al, Oncogene 18 (1999) 1711-1721. In one embodiment the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the invention is between heavy chain variable domain position 105 and light chain variable domain position 43 (numbering always according to EU index of Kabat). In an embodiment single chain Fab fragment without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragments are preferred. The full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE class (or isotypes) and, in the case of IgG and IgA, their subclasses (or subtypes). In a preferred embodiment, an full length antibody of the invention and thus a monovalent antigen binding protein of the invention has a constant domain structure of an IgG class antibody.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.
The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g.,
Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.
The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody." See, e.g., Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Bruggemann, M., et al, Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation).
The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
The "variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding. CDR and FR regions are determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
As used herein, the term "binding" or "specifically binding" refers to the binding of the multispecific antigen binding protein to an epitope of the respective antigen in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE- Healthcare Uppsala, Sweden) with purified wild-type antigen. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), kD (dissociation constant), and KD (kD/ka). Binding or specifically binding means a binding affinity (KD) of 10~8 mo 1/1 or less, preferably 10~9 M to 10~13 mo 1/1. Thus, a multispecific antigen binding protein to the invention is specifically binding to each antigen for which it is specific with a binding affinity (KD) of 10"8 mol/1 or less, preferably 10"9 M to 10"13 mol/1.
The term "epitope" includes any polypeptide determinant capable of specific binding to a monovalent antigen binding proteins. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by a monovalent antigen binding protein.
In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
In a further embodiment the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgGl subclass, or of human IgGl subclass with the mutations L234A and L235A.
In a further embodiment the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG2 subclass. In a further embodiment the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG3 subclass.
In a further embodiment the monovalent antigen binding protein under A) is characterized in that said full length antibody is of human IgG4 subclass or, of human IgG4 subclass with the additional mutations S228P and L235E (also named
IgG4 SPLE).
The term "constant region" as used within the current applications denotes the sum of the domains of an antibody other than the variable region. The constant region is not involved directly in binding of an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided in the classes (also named isotypes): IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (also named isotypes), such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) which can be found in all five antibody classes are called κ (kappa) and λ (lambda).
The term "constant region derived from human origin" as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al, Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788).
While antibodies of the IgG4 subclass show reduced Fc receptor (FcyRIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238,
Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which, if altered, provide also reduced Fc receptor binding (Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al, FASEB J. 9 (1995) 115-119; Morgan, A., et al, Immunology 86 (1995) 319-324; EP 0 307 434).
In one embodiment an antibody according to the invention has a reduced FcR binding compared to an IgGl antibody and the full length parent antibody is in regard to FcR binding of IgG4 subclass or of IgGl or IgG2 subclass with a mutation in S228, L234, L235 and/or D265, and/ or contains the PVA236 mutation. In one embodiment the mutations in the full length parent antibody are S228P, L234A, L235A, L235E and/or PVA236. In another embodiment the mutations in the full length parent antibody are in IgG4 S228P and L235E and in IgGl L234A and L235A. The constant region of an antibody is directly involved in ADCC (antibody- dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by binding of complement factor Clq to the constant region of most IgG antibody subclasses. Binding of Clq to an antibody is caused by defined protein-protein interactions at the so called binding site. Such constant region binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al, J. Immunol. 127 (1981) 2555-2560; Bunkhouse, R. and Cobra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R., et al, Nature 288 (1980) 338-344; Thomason, J.E., et al, Mol. Immunol. 37 (2000) 995-1004; Idiocies, E.E., et al, J. Immunol. 164 (2000) 4178-4184; Hearer, M., et al, J. Virol. 75 (2001) 12161-12168; Morgan, A., et al, Immunology 86 (1995)
319-324; and EP 0 307 434. Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is measured preferably by the treatment of a preparation of antigen expressing cells with an antibody according to the invention in the presence of effector cells such as freshly isolated PBMC or purified effector cells from buffy coats, like monocytes or natural killer (NK) cells or a permanently growing NK cell line.
The term "complement-dependent cytotoxicity (CDC)" denotes a process initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. Binding of Clq to an antibody is caused by defined protein-protein interactions at the so called binding site. Such Fc part binding sites are known in the state of the art (see above). Such Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclass IgGl, IgG2, and IgG3 usually show complement activation including Clq and C3 binding, whereas IgG4 does not activate the complement system and does not bind Clq and/or C3. Cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and US 6,602,684. IgGl type antibodies, the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody dependent cellular cytotoxicity (ADCC) (Lifely, M.R., et al, Glycobiology 5 (1995) 813-822; Jefferis, R., et al, Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, S., L., Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al, Nature Biotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpression in Chinese hamster ovary (CHO) cells of B(l,4)-N- acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase catalyzing the formation of bisected oligosaccharides, significantly increases the in vitro ADCC activity of antibodies. Alterations in the composition of the Asn297 carbohydrate or its elimination affect also binding to FcyR and Clq (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al, Biotechnol. Bioeng. 74 (2001) 288-294; Mimura, Y., et al, J. Biol. Chem. 276 (2001) 45539-45547; Radaev, S., et al, J. Biol. Chem. 276 (2001) 16478-16483; Shields, R.L., et al, J. Biol. Chem.
276 (2001) 6591-6604; Shields, R.L., et al, J. Biol. Chem. 277 (2002) 26733- 26740; Simmons, L.C., et al, J. Immunol. Methods 263 (2002) 133-147).
In one aspect of the invention the monovalent antigen binding protein under A) is characterized in that the modified heavy chains of a) and b) are of IgGl isotype, and the antigen binding protein is afucosylated with an the amount of fucose of
80 % or less of the total amount of oligosaccharides (sugars) at Asn297.
In one embodiment the antigen binding protein is afucosylated with an the amount of fucose of 65 % to 5 % of the total amount of oligosaccharides (sugars) at Asn297. The term "afucosylated antigen binding protein" refers to an antigen binding protein under A) of IgGl or IgG3 isotype (preferably of IgGl isotype) with an altered pattern of glycosylation in the Fc region at Asn297 having a reduced level of fucose residues. Glycosylation of human IgGl or IgG3 occurs at Asn297 as core fucosylated bianntennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues. These structures are designated as GO, Gl (al,6 or al,3) or G2 glycan residues, depending from the amount of terminal Gal residues (Raju, T.S., BioProcess Int. 1 (2003) 44-53). CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F.H., Glycoconjugate J. 14 (1997) 201-207. Antibodies which are recombinantely expressed in non glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85 %. It should be understood that the term an afucosylated antibody or antigen binding protein as used herein includes an antibody or antigen binding protein having no fucose in its glycosylation pattern. It is commonly known that typical glycosylated residue position in an antibody is the asparagine at position 297 according to the EU numbering system ("Asn297").
Thus an afucosylated antigen binding protein under A) means an antibody of IgGl or IgG3 isotype (preferably of IgGl isotype) wherein the amount of fucose is 80 % or less (e.g. of 80 % to 1 %) of the total amount of oligosaccharides (sugars) at Asn297 (which means that at least 20 % or more of the oligosaccharides of the Fc region at Asn297 are afucosylated). In one embodiment the amount of fucose is
65 % or less (e.g. of 65 % to 1 %), in one embodiment from 65 % to 5 %, in one embodiment from 40 % to 20 % of the oligosaccharides of the Fc region at Asn297. According to the invention "amount of fucose" means the amount of said oligosaccharide (fucose) within the oligosaccharide (sugar) chain at Asn297, related to the sum of all oligosaccharides (sugars) attached to Asn 297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry and calculated as average value (for a detailed procedure to determine the amount of fucose, see e.g. WO 2008/077546). Furthermore in one embodiment, the oligosaccharides of the Fc region are bisected. The afucosylated antibody or antigen binding protein according to the invention can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity in an amount sufficient to partially fucosylate the oligosaccharides in the Fc region. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively al,6-fucosyltransferase activity of the host cell can be decreased or eliminated according to US 6,946,292 to generate glycomodified host cells. The amount of antibody fucosylation can be predetermined e.g. either by fermentation conditions (e.g. fermentation time) or by combination of at least two antibodies with different fucosylation amount. Such afucosylated antigen binding proteins and respective glycoengineering methods are described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P., et al, Nature Biotechnol. 17 (1999) 176-180, WO 99/54342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2005/011735, WO 2005/027966, WO 97/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739. These glycoengineered antigen binding proteins according to the invention have an increased ADCC (compared to the parent antigen binding proteins). Other glycoengineering methods yielding afucosylated antigen binding proteins according to the invention are described e.g. in Niwa, R.. et al, J. Immunol. Methods 306 (2005) 151-160; Shinkawa, T., et al, J. Biol. Chem, 278 (2003) 3466-3473; WO 03/055993 or US 2005/0249722.
The "EU numbering system" or "EU index (according to Kabat)" is generally used when referring to a residue or position in an immunoglobulin heavy chain constant region (e.g., the EU index is reported in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) expressly incorporated herein by reference).
The term "the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell" denotes that the sugar chain at Asn297 of the full length parent antibody according to the invention has the same structure and sugar residue sequence except for the fucose residue as those of the same antibody expressed in unmodified CHO cells, e.g. as those reported in WO 2006/103100. The term "NGNA" as used within this application denotes the sugar residue
N-glycolylneuraminic acid.
The antibody according to the invention is produced by recombinant means. Thus, one aspect of the current invention is a nucleic acid encoding the antibody according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the antibodies as aforementioned in a host cell, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al, Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880. The multispecific antigen binding proteins according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA and RNA encoding the multispecific antigen binding proteins is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant multispecific antigen binding proteins in the host cells.
Amino acid sequence variants (or mutants) of the multispecific antigen binding proteins are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
The term "host cell" as used in the current application denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention. In one embodiment HEK293 cells and CHO cells are used as host cells.
As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
Expression in NS0 cells is described by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al, J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
Purification of multispecific antigen binding proteins is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins (e.g. byproducts) by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art (see Ausubel, F., et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed- mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102). An example of a purification is described in Example 1 and the corresponding Figures 3 to 8.
One aspect of the invention is a pharmaceutical composition comprising an multispecific antigen binding protein according to the invention. Another aspect of the invention is the use of a multispecific antigen binding protein according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising a multispecific antigen binding proteins according to the invention. In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing a multispecific antigen binding proteins according to the present invention, formulated together with a pharmaceutical carrier. One embodiment of the invention is the multispecific antigen binding protein according to the invention for the treatment of cancer.
Another aspect of the invention is said pharmaceutical composition for the treatment of cancer.
Another aspect of the invention is the use of a multispecific antigen binding proteins according to the invention for the manufacture of a medicament for the treatment of cancer.
Another aspect of the invention is method of treatment of patient suffering from cancer by administering a multispecific antigen binding proteins according to the invention to a patient in the need of such treatment. As used herein, "pharmaceutical 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 (e.g. by injection or infusion). A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
The term cancer as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
The term "transformation" as used herein refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection is carried out e.g. by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 546. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride as described by Cohen, F.N, et al, PNAS. 69 (1972) 7110.
As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.
A "vector" is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.
An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product. The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Sequence Listing
SEQ ID NO: 1 VEGF MoAb heavy chain 1 (hole, Avastin CHl-Vh)
SEQ ID NO: 2 MoAb heavy chain 2 (knob, Avastin Ck-Vl)
SEQ ID NO: 3 Ang2i-LC06-CHl-VH
SEQ ID NO: 4 Ang2i-LC06-Ck-Vl
SEQ ID NO: 5 Ang2i-LC06-VH
SEQ ID NO: 6 Ang2i-LC06-Vl
SEQ ID NO: 7 scFab-Ang2i-LC06 (VI- Vh)
SEQ ID NO: 8 HC1 (HOLE): SSHC2-AVA-VHCH1-N-LC-LC06
SEQ ID NO:9 HC2 (KNOB): SSKHC1-AVALC-N-VHCHLC06
SEQ ID NO: 10 HC1 (Hole): SSHC2-Ava-VHCHl-N-VL-LC06
SEQ ID NO: 11 HC2 (Knob): SSHCl-AvaLC-N-VH-LC06
SEQ ID NO: 12 HC1 (Hole): SSHC2-Ava-VHCH1
SEQ ID NO: 13 HC2 (Knob): SSKHCl-AvaLC-scFabLC06
SEQ ID NO: 14 HC1: XAng2i 6Q-VH-Ckappa-FcSShole
SEQ ID NO: 15 HC2: VL-Ang2-HC-knob_GS_scFab-Ava
In the following, embodiments of the invention are listed:
A multispecific antigen binding protein, comprising
A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
B) an antigen binding peptide which specifically binds to a second antigen, wherein the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus or N-terminus of one or both heavy chains under A).
The multispecific antigen binding protein according to embodiment 1 characterized in being a bispecific, bivalent antibody.
The multispecific antigen binding protein according to any one of claims 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody.
The multispecific antigen binding protein according to any one of embodiments 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody. The multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a Fv fragment, Fab fragment, a scFv fragment, and a scFab fragment. The multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a Fv fragment and Fab fragment which are fused via two peptide connectors to the C-terminus or N-terminus of both heavy chains under A). The multispecific antigen binding protein according to any one of embodiments 1 to 4 characterized in that the antigen binding peptide is selected from the group of a scFv fragment and scFab fragment which are fused via one peptide connector to the C-terminus or N-terminus of both heavy chains under A). The multispecific antigen binding protein according to any one of embodiments 5 or 6 characterized in that the antigen binding peptide is a Fv fragment. The multispecific antigen binding protein according to any one of embodiments 5 or 6 characterized in that the antigen binding peptide is a Fab fragment. The multispecific antigen binding protein according to any one of embodiments 5 or 7 characterized in that the antigen binding peptide is a scFv fragment. The multispecific antigen binding protein according to any one of embodiments 5 or 7 characterized in that the antigen binding peptide is a scFab fragment. The multispecific antigen binding protein according to any one of embodiments 1 to 11 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the N-terminus of one or both heavy chains under A). The multispecific antigen binding protein according to any one of embodiments 1 to 11 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus of one or both heavy chains under A). The multispecific antigen binding protein according to embodiment 1 characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and
b) the amino acid sequence of SEQ ID NO:9;
or a) the amino acid sequence of SEQ ID NO: 10; and
b) the amino acid sequence of SEQ ID NO:l 1;
or a) the amino acid sequence of SEQ ID NO: 12; and
b) the amino acid sequence of SEQ ID NO: 13;
or a) the amino acid sequence of SEQ ID NO: 14; and
b) the amino acid sequence of SEQ ID NO: 15;. The multispecific antigen binding protein to any one of embodiments 1 to 13 characterized in that the heavy chains under A) are of human IgG isotype. The multispecific antigen binding protein according to embodiment 15 characterized in that the heavy chains under A) are of human IgGl or IgG4 isotype. A method for the preparation of a multispecific antigen binding protein according to the invention comprising the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding a multispecific antigen binding protein according to embodiments 1 to 16 b) culturing the host cell under conditions that allow synthesis of said multispecific antigen binding protein molecule; and c) recovering said multispecific antigen binding protein molecule from said culture. 18. Nucleic acid encoding the multispecific antigen binding protein embodiments
1 to 16.
19. Vectors comprising nucleic acid according to embodiment 18 encoding the multispecific antigen binding protein embodiments 1 to 16.
20. A host cell comprising vectors according to embodiment 19. 21. A composition, preferably a pharmaceutical or a diagnostic composition of a multispecific antigen binding protein embodiments 1 to 16.
22. A pharmaceutical composition comprising a multispecific antigen binding protein according to embodiments 1 to 16 and at least one pharmaceutically acceptable excipient. 23. Use of a multispecific antigen binding protein according to embodiments 1 to
16 for the manufacture of a medicament for the treatment of a disease, preferably cancer or an inflammatory disease.
24. A multispecific antigen binding protein according to embodiments 1 to 16 for for use in the treatment of a disease, preferably cancer or an inflammatory disease.
25. A method for the treatment of a patient ( preferably suffering from cancer or an inflammatory disease) in need of therapy, characterized by administering to the patient a therapeutically effective amount of a multispecific antigen binding protein binding protein according to embodiments 1 to 16. Experimental Procedure Examples
Materials & Methods
Recombinant DNA techniques Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.
DNA and protein sequence analysis and sequence data management General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242. Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et al, PNAS 63 (1969) 78-85; Kabat, E.A., et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH
Publication No 91-3242). The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NTI Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration. DNA sequencing
DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
Gene synthesis
Desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis.
The gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene Segments coding "knobs-into-hole" monovalent antibody heavy chain carrying a T366W mutation in the CH3 domain with a C-terminal or N- terminal antigen binding peptide linked by peptide connector(s) as well as "knobs- into-hole" heavy chain carrying T366S, L368A and Y407V mutations with a C- terminal or N-terminal antigen binding peptide linked by peptide connector(s) were synthesized with 5 '-BamHI and 3 '-Xbal restriction sites. In a similar manner, DNA sequences coding "knobs-into-hole" heavy chain carrying S354C and T366W mutations in the CH3 domain with a C-terminal or N-terminal antigen binding peptide linked by peptide connector(s) as well as "knobs-into-hole" heavy chain carrying Y349C, T366S, L368A and Y407V mutations with a C-terminal or N- terminal antigen binding peptide linked by peptide connector(s) were prepared by gene synthesis with flanking BamHI and Xbal restriction sites. All constructs were designed with a 5 '-end DNA sequence coding for a leader peptide (MGWSCIILFLVATATGVHS), which targets proteins for secretion in eukaryotic cells. Construction of the expression plasmids
The following expression vector was used for the construction of all heavy and light chain fusion protein encoding expression plasmids. The vector is composed of the following elements: a hygromycin resistance gene as a selection marker,
- an origin of replication, oriP, of Epstein-Barr virus (EBV),
an origin of replication from the vector pUC 18 which allows replication of this plasmid in E. coli
a beta-lactamase gene which confers ampicillin resistance in E. coli, the immediate early enhancer and promoter from the human cytomegalovirus (HCMV),
the human 1 -immunoglobulin polyadenylation ("poly A") signal sequence, and
unique BamHI and Xbal restriction sites.
The immunoglobulin fusion genes comprising the heavy chain constucts as well as "knobs-into-hole" constructs with a C-terminal or N-terminal antigen binding peptide linked by peptide connector(s) were prepared by gene synthesis and cloned into pGA18 (ampR) plasmids as described. The pG18 (ampR) plasmids carrying the synthesized DNA segments and the Roche expression vector were digested with BamHI and Xbal restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis. Purified heavy and light chain coding DNA segments were then ligated to the isolated Roche expression vector BamHI/Xbal fragment resulting in the final expression vectors. The final expression vectors were transformed into E. coli cells, expression plasmid DNA was isolated (Miniprep) and subjected to restriction enzyme analysis and DNA sequencing. Correct clones were grown in 150 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep) and sequence integrity confirmed by DNA sequencing.
Cell culture techniques Standard cell culture techniques were used as described in Current Protocols in
Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott- Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.
Transient transfections in HEK293-F system
Antibodies were generated by transient transfection of the two plasmids encoding the heavy or modified heavy chain one, respectively and the corresponding heavy chain two using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serumfree FreeStyle 293 expression medium (Invitrogen) were transfected with a mix of the two respective expression plasmids and 293fectin or fectin (Invitrogen). For e.g. 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8 % C02. The day after the cells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modified heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM + 1.2 mL 293 fectin or fectin (2 μΙ/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.
Protein determination
The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et. al, Protein Science, 1995, 4, 2411-1423.
Antibody concentration determination in supernatants
The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with Protein A Agarose-beads (Roche). 60
Protein A Agarose beads are washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1 - 15 mL cell culture supernatant are applied to the Protein A Agarose beads pre-equilibrated in TBS- NP40. After incubation for at 1 h at room temperature the beads are washed on an Ultrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2x phosphate buffered saline (2xPBS, Roche) and briefly four times with 0.5 mL 100 mM Na-citrate pH 5,0. Bound antibody is eluted by addition of 35 μΐ NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample is combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated for 10 min at 70°C. Consequently, 20 μΐ are applied to an 4-12 % NuPAGE® Bis-Tris
SDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.
The concentration of antibodies and derivatives in cell culture supernatants was measured by Protein A-HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to Protein A were applied to a HiTrap Protein A column (GE Healthcare) in 50 mM K2HP04, 300 mM NaCl, pH 7.3 and eluted from the matrix with 550 mM acetic acid, pH 2.5 on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. A purified standard IgGl antibody served as a standard.
Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche) were coated with 100 μΕΛνεΙΙ biotinylated anti-human IgG capture molecule F(ab')2<h-Fcgamma> BI (Dianova) at 0.1 μg/mL for 1 h at room temperature or alternatively over night at 4°C and subsequently washed three times with 200 μΕΛνεΙΙ PBS, 0.05 % Tween (PBST, Sigma). 100 μΕΛνεΙΙ of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 h on a microtiterplate shaker at room temperature. The wells were washed three times with 200 μΙ,ΛνεΙΙ PBST and bound antibody was detected with 100 μΐ F(ab')2<hFcgamma>POD (Dianova) at 0.1 μg/mL as detection antibody for 1-2 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 200 μίΛνεΙΙ PBST and the bound detection antibody was detected by addition of 100 ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
Protein purification
Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS. Elution of antibodies was achieved at acidic pH followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM Histidine, 140 mM NaCl pH 6.0. Monomeric antibody fractions were pooled, concentrated if required using e.g. a
MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator and stored at -80 °C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography, mass spectrometry and Endotoxin determination (see Figures 3 and 4). SDS-PAGE
The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 4 - 20 % NuPAGE® Novex® TRIS- Glycine Pre-Cast gels and a Novex® TRIS-Glycine SDS running buffer were used. Reducing of samples was achieved by adding NuPAGE® sample reducing agent prior to running the gel.
Chip-based CE-SDS
Traditionally, SDS-PAGE (sodium dodecylsulfate-based polyacrylamide gel electrophoresis) is used as a method to determine the approximate molecular weight of denatured proteins. Modern implementations of the same separation principle involve capillary electrophoresis with subsequent fluorescence-based detection of the separated proteins. The result is an approximate molecular weight of the denatured proteins, either with intact disulfide bonds (non-reduced) or after disruption of disulfide bonds (reduced). Additionally, CE-SDS allows quantification of the individual components.
Analytical size exclusion chromatography
Size exclusion chromatography for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly,
Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2P04/K2HP04, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2 x PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151 - 1901 served as a standard, (see e.g. Figure 4).
SEC-MALLS
SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) was used to determine the approximate molecular weight of proteins or protein conjugates in solution. According to the light scattering theory, the intensity of the scattered light is proportional to the molar concentration of the macromolecules in solution. SEC-MALLS is based on a separation of proteins according to their size (hydrodynamic radius) via SEC chromatography, followed by concentration- and scattered light-sensitive detectors. Concentration-sensitive detectors can be e.g. UV absorption detectors or differential refractive index (RI) detectors. SEC-MALLS typically gives rise to molecular weight estimates with an accuracy that allows clear discrimination between monomers, dimers, trimers etc., provided the SEC separation is sufficient.
In the current work, the following instrumentation was used: Dionex Ultimate 3000 HPLC; column: Superose6 10/300 (GE Healthcare); eluent: lx PBS; detectors: OptiLab REX (Wyatt Inc., Dernbach), MiniDawn Treos (Wyatt Inc., Dernbach).
Mass spectrometry
The total deglycosylated mass of antibodieswas determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 μg purified antibodies were deglycosylated with 50 mU N-Glycosidase F (PNGaseF,
ProZyme) in 100 mM KH2P04/K2HP04, pH 7 at 37°C for 12-24 h at a protein concentration of up to 2 mg/ml and subsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare). The mass of the respective heavy and light chains was determined by ESI-MS after deglycosylation and reduction. In brief, 50 μg antibody in 115 μΐ were incubated with 60 μΐ 1M TCEP and 50 μΐ 8 M Guanidine- hydrochloride subsequently desalted. The total mass and the mass of the reduced heavy and light chains was determined via ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source.
VEGF binding ELISA
The binding properties of the antibodies was evaluated in an ELISA assay with full-length VEGF165-His protein (R&D Systems) (Figure 5). For this sake Falcon polystyrene clear enhanced microtiter plates were coated with 100 μΐ 2 μg/mL recombinant human VEGF 165 (R&D Systems) in PBS for 2 h at room temperature or over night at 4°C. The wells were washed three times with 300 μΐ PBST (0,2% Tween 20) and blocked with 200 μΐ 2 % BSA 0,1 % Tween 20 for 30 min at room temperature and subsequently washed three times with 300μ1 PBST. 100 μΕΛνεΙΙ of a dilution series (40 pM-0.01 pM) of purified antibody in PBS (Sigma) was added to the wells and incubated for 1 h on a microtiterplate shaker at room temperature. The wells were washed three times with 300μ1 PBST (0,2 % Tween 20) and bound antibody was detected with 100 μΕΛνεΙΙ 0.1 μg/ml F(ab') <hFcgamma>POD (Immuno research) in 2 % BSA 0, 1% Tween 20 as detection antibody for 1 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 300 μΕΛνεΙΙ PBST and the bound detection antibody was detected by addition of 100 μΐ, ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm). ANG-2 binding ELISA
The binding properties of the antibodies was evaluated in an ELISA assay with full-length Angiopoietin-2-His protein (R&D Systems) (Figure 6a). For this sake Falcon polystyrene clear enhanced microtiter plates were coated with 100 μΐ 1 μg/mL recombinant human Angiopoietin-2 (R&D Systems, carrier-free) in PBS for 2 h at room temperature or over night at 4° C. The wells were washed three times with 300μ1 PBST (0,2% Tween 20) and blocked with 200 μΐ 2% BSA 0,1% Tween 20 for 30 min at room temperature and subsequently washed three times with 300μ1 PBST. 100 μίΛνεΙΙ of a dilution series (40pM-0.01 pM) of purified antibody in PBS (Sigma) was added to the wells and incubated for 1 h on a microtiterplate shaker at room temperature. The wells were washed three times with 300μ1 PBST (0,2 % Tween 20) and bound antibody was detected with 100 μΙ,ΛνεΙΙ 0.1 μg/ml F(ab') <hk>POD (Biozol Cat.No. 206005) in 2 % BSA 0,1 % Tween 20 as detection antibody for 1 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 300 μΙ,ΛνεΙΙ PBST and the bound detection antibody was detected by addition of 100 μΐ, ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
Comparative binding to ANG-1 and ANG-2 (ANG-1 and ANG-2 binding ELISA)
The binding properties of antibodies were evaluated in an ELISA assay with full- length Angiopoietin-2-His protein (R&D Systems #623-AN/CF or in house produced material) or Angiopoietin-l-His (R&D systems #923 -AN). Therefore 96 well plates (Falcon polystyrene clear enhanced microtiter plates or Nunc Maxisorb) were coated with 100 μΐ 1 μg/mL recombinant human Angiopoietin-1 or
Angiopoietin-2 (carrier-free) in PBS (Sigma) for 2 h at room temperature or over night at 4°C. The wells were washed three times with 300μ1 PBST (0,2 % Tween 20) and blocked with 200 μΐ 2 % BSA 0,1 % Tween 20 for 30 min at room temperature and subsequently washed three times with 300μ1 PBST. 100 μΙ,ΛνεΙΙ of a dilution series (40 pM-0.01 pM) of purified test antibody in PBS was added to the wells and incubated for 1 h on a microtiterplate shaker at room temperature. The wells were washed three times with 300μ1 PBST (0,2% Tween 20) and bound antibody was detected with 100 μΕ/well 0.1 μg/ml F(ab') <hk>POD (Biozol Cat.No. 206005) in 2% BSA 0,1% Tween 20 as detection antibody for 1 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 300 μΙ,ΛνεΙΙ PBST and the bound detection antibody was detected by addition of 100 μί ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm). Inhibition of huANG-2 binding to Tie-2 (ELISA)
The interaction ELISA was performed on 384 well microtiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After each incubation step plates were washed 3 times with PBST. ELISA plates were coated with 0.5 μg/ml Tie-2 protein (R&D Systems, UK, Cat.No.313-TI) for at least 2 hours (h). Thereafter the wells were blocked with PBS supplemented with 0.2 % Tween-20 and 2 % BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions of purified antibodies in PBS were incubated together with 0.2 μg/ml huAngiopoietin-2 (R&D Systems, UK, Cat.No. 623-AN) for 1 h at RT. After washing a mixture of 0.5 μg/ml biotinylated anti- Angiopoietin-2 clone BAM0981 (R&D Systems, UK) and 1 :3000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat.No.11089153001) was added for 1 h. Thereafter the plates were washed 6 times with PBST. Plates were developed with freshly prepared ABTS reagent (Roche Diagnostics GmbH, DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT. Absorbance was measured at 405 nm.
ANG-2 binding BIACORE
Binding of the antibodies to the antigen e.g. human ANG-2 were investigated by surface plasmon resonance using a BIACORE T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements goat<hIgG- Fcy> polyclonal antibodies were immobilized on a CM5 chip via amine coupling for presentation of the antibodies against human ANG-2 (Figure 6B). Binding was measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005 % Tween 20, ph 7.4), 25°C. Purified ANG-2-His (R&D systems or in house purified) was added in various concentrations between 6,25 nM and 200 nM in solution. Association was measured by an ANG-2-injection of 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3 minutes and a KD value was estimated using a 1 : 1 Langmuir binding model. Due to heterogenity of the ANG-2 preparation no 1 : 1 binding could be observed; KD values are thus only relative estimations. Negative control data (e.g. buffer curves) were subtracted from sample curves for correction of system intrinsic baseline drift and for noise signal reduction. Biacore T100 Evaluation Software version 1.1.1 was used for analysis of sensorgrams and for calculation of affinity data. Alternatively, Ang-2 could be captured with a capture level of 2000-1700 RU via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen No. 34660) that was immobilized on a CM5 chip via amine coupling (BSA-free) (see below).
VEGF binding: Kinetic characterization of VEGF binding at 37°C by surface plasmon resonance (Biacore)
In order to further corroborate the ELISA findings the binding of antibodies to VEGF was quantitatively analyzed using surface plasmon resonance technology on a Biacore T100 instrument according to the following protocol and analyzed using the T100 software package: Briefly <VEGF-ANG2> antibodies were captured on a CM5-Chip via binding to a Goat Anti Human IgG (JIR 109-005-098). The capture antibody was immobilized by amino coupling using standard amino coupling as follows: HBS-N buffer served as running buffer, activation was done by mixture of
EDC/NHS with the aim for a ligand density of 700 RU. The Capture- Antibody was diluted in coupling buffer NaAc, pH 5.0, c = 2 μg/mL, finally still activated carboxyl groups were blocked by injection of 1 M Ethanolamine. Capturing of Mabs <VEGF-ANG2> antibodies was done at a flow of 5 μΕ/ηιίη and c(Mabs<VEGF>) = 10 nM, diluted with running buffer + 1 mg/mL BSA; a capture level of approx. 30 RU should be reached. rhVEGF (rhVEGF, R&D-Systems Cat.- No, 293-VE) was used as analyte. The kinetic characterization of VEGF binding to <VEGF-ANG2> antibodies was performed at 37°C in PBS + 0.005 % (v/v) Tween20 as running buffer. The sample was injected with a flow of 50 μΕ/ηιίη and an association of time 80 sec. and a dissociation time of 1200 sec with a concentration series of rhVEGF from 300 - 0.29 nM. Regeneration of free capture antibody surface was performed with 10 mM Glycin pH 1.5 and a contact time of 60 sec after each analyte cycle. Kinetic constants were calculated by using the usual double referencing method (control reference: binding of rhVEGF to capture molecule Goat Anti Human IgG, blanks on the measuring flow cell, rhVEGF concentration "0", Model: Langmuir binding 1 : 1, (Rmax set to local because of capture molecule binding). Figure 11 shows a schematic view of the Biacore assay.
Generation of HEK293-Tie2 cell line
In order to determine the interference of Angiopoietin-2 antibodies with ANGPT2 (ANG2) stimulated Tie2 phosphorylation and binding of ANGPT2 to Tie2 on cells a recombinant HEK293-Tie cell line was generated. Briefly, a pcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2) coding for full-length human Tie2 (SEQ ID 108) under control of a CMV promoter and a Neomycin resistance marker was transfected using Fugene (Roche Applied Science) as transfection reagent into HEK293 cells (ATCC) and resistant cells were selected in DMEM 10 % FCS, 500μg/ml G418. Individual clones were isolated via a cloning cylinder, and subsequently analyzed for Tie2 expression by FACS. Clone 22 was identified as clone with high and stable Tie2 expression even in the absence of G418 (HEK293- Tie2 clone22). HEK293-Tie2 clone22 was subsequently used for cellular assays: ANGPT2 induced Tie2 phosphorylation and ANGPT2 cellular ligand binding assay. ANGPT2 induced Tie2 phosphorylation assay
Inhibition of ANGPT2 (ANG2) induced Tie2 phosphorylation by <VEGF-ANG2> antibodies was measured according to the following assay principle. HEK293-Tie2 clone22 was stimulated with ANGPT2 for 5 minutes in the absence or presence of ANGPT2 antibody and P-Tie2 was quantified by a sandwich ELISA. Briefly,
2x105 HEK293-Tie2 clone 22 cells per well were grown over night on a Poly-D- Lysine coated 96 well- microtiter plate in ΙΟΟμΙ DMEM, 10% FCS, 500 μ^πιΐ Geneticin. The next day a titration row of ANGPT2 antibodies was prepared in a microtiter plate (4-fold concentrated, 75 μΐ final volume/well, duplicates) and mixed with 75μ1 of an ANGPT2 (R&D systems # 623-AN] dilution (3.2 μ^πιΐ as 4-fold concentrated solution). Antibodies and ANGPT2 were pre-incubated for 15 min at room temperature. 100 μΐ of the mix were added to the HEK293-Tie2 clone 22 cells (pre-incubated for 5 min with 1 mM NaV304, Sigma #S6508) and incubated for 5 min at 37°C. Subsequently, cells were washed with 200μ1 ice-cold PBS + ImM NaV304 per well and lysed by addition of 120μ1 lysis buffer (20 mM
Tris, pH 8.0, 137 mM NaCl, 1 % NP-40, 10 % glycerol, 2mM EDTA, I mM NaV304, 1 mM PMSF and 10 μ^ιηΐ Aprotinin) per well on ice. Cells were lysed for 30 min at 4°C on a microtiter plate shaker and 100 μΐ lysate were transferred directly into a p-Tie2 ELISA microtiter plate (R&D Systems, R&D #DY990) without previous centrifugation and without total protein determination. P-Tie2 amounts were quantified according to the manufacturer's instructions and IC50 values for inhibition were determined using XLfit4 analysis plug-in for Excel (Dose-response one site, model 205). IC50 values can be compared within on experiment but might vary from experiment to experiment. VEGF induced HUVEC proliferation assay
VEGF induced HUVEC (Human Umbilical Vein Endothelial Cells, Promocell #C- 12200) proliferation was chosen to measure the cellular function of VEGF antibodies. Briefly, 5000 HUVEC cells (low passage number, <5 passages) per 96 well were incubated in ΙΟΟμΙ starvation medium (EBM-2 Endothelial basal medium 2, Promocell # C-22211, 0.5% FCS, Penicilline/Streptomycine) in a collagen I-coated BD Biocoat Collagen I 96-well microtiter plate (BD #354407 / 35640 over night. Varying concentrations of antibody were mixed with rhVEGF (30 ngl/ml final concentration, BD # 354107) and pre-incubated for 15 minutes at room temperature. Subsequently, the mix was added to the HUVEC cells and they were incubated for 72 h at 37°C, 5% C02. On the day of analysis the plate was equilibrated to room temperature for 30 min and cell viability/proliferation was determined using the CellTiter-GloTM Luminescent Cell Viability Assay kit according to the manual (Promega, # G7571/2/3). Luminescence was determined in a spectrophotometer. Mouse cornea micropocket angiogenesis assay
8 to 10 weeks old female Balb/c mice were purchased from Charles River, Sulzfeld, Germany. The protocol was modified according to the method described by Rogers et al. (2007). Briefly, micropockets with a width of about 500 μιη were prepared under a microscope at approximately 1 mm from the limbus to the top of the cornea using a surgical blade and sharp tweezers in the anesthetized mouse. The disc (Nylaflo®, Pall Corporation, Michigan) with a diameter of 0.6 mm was implanted and the surface of the implantation area was smoothened. Discs were incubated in corresponding growth factor or in vehicle for at least 30 min. After 3, 5 and 7 days, eyes were photographed and vascular response was measured. The assay was quantified by calculating the percentage of the area of new vessels per total area of the cornea.
Design of bispecific, bivalent antigen binding proteins according to the invention
The bispecific, bivalent antigen binding proteins according to the invention binding to VEGF (VEGF-A) and ANG-2 (Angiopoietin-2) comprise a first antigen-binding site that binds to VEGF and a second antigen-binding site that binds to ANG-2. As first antigen-binding site binding to VEGF, e.g. the heavy chain variable domain of the light chain variable domains from the anti-VEGF antibodies <VEGF>bevacizumab (see e.g. WO2010/040508 which incorporated by reference). As second antigen-binding site comprises the heavy chain variable domains and the light chain variable domains from the anti-ANG-2 antibodies <ANG-2> Ang2s_R3_LC03, <ANG-2>Ang2i_LC06, <ANG-2>Ang2i_LC07, <ANG-2> Ang2k_LC08, <ANG-2> Ang2s_LC09, <ANG-2> Ang2i_LC10, or <ANG-2> Ang2k_LCl l, preferably from <ANG-2>Ang2i_LC06, or <ANG-2> Ang2k_LC08 bevacizumab ( see e.g. WO 2010/040508 which incorporated by reference).
To generate agents that combine features of both antibodies, novel bispecific monovalent antibody-derived protein entities were constructed. The basis for this novel bispecific, bivalent antigen binding proteins are monovalent antibodies (MoAb) as described e.g. in US 2004/0033561 and PCT Application PCT/EP2012/053119. They consist of 2 different heavy chains: i) a) a first heavy chain of an full length antibody; and
b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
In i) heavy chain 1 (HC1) is a typical IgG like heavy chain consisting of VH-CH1- CH2-CH3 domains. Heavy chain 2 (HC2) consists of VL-CL-CH2-CH3 domains. In ii) heavy chain 1 (HC1) is a typical IgG like heavy chain consisting of VH-CL- CH2-CH3 domains. Heavy chain 2 (HC2) consists of VL-CH 1 -CH2-CH3 domains
The construction of a bispecific, bivalent antigen binding protein according to the invention (BiMoMAb) is based on N-, or C-terminal fusions of recombinant Fv binding, single-chain Fv binding, single chain Fab binding and Fab binding domains of a second specificity, which are connected via recombinant protein fusion technologies to the monovalent antibody.
By gene synthesis and recombinant molecular biology techniques, the heavy chain Fab domain (VH-CH1) and the light chain Fab domain (VL-CL) of the respective antibody were linked by a glycine serine (G4S)3 or (G4S)4 single-chain-linker to give a single chain Fv (scFab), which was attached to the C- terminus of the other antibody heavy chain using e.g. a (G4S)3-connector.
In addition, cysteine residues were introduced in the VH (including Kabat position 44,) and VL (including Kabat position 100) domain of the scFv binding to ANG-2 or VEGF as described earlier (e.g. WO 94/029350; Reiter et al, Nature Biotechnology (1996) 1239-1245; Young et al, FEBS Letters (1995) 135-139; or Rajagopal et al, Protein Eng. (1997) 1453-59).
All these molecules were recombinantly produced, purified and characterized and protein expression, stability and biological activity was evaluated. A summary of the bispecific antibody designs that were applied to generate monovalent bispecific <VEGF-ANG-2>, <ANG-2-VEGF> antibodies a is given in Table la and b. For this study, we use the term 'BiMoMAb' (bispecific, bivalent antigen protein based on monovalent monoclonal antibody) to describe the various bispecific protein entities.
In order to obtain the bispecific antigen binding proteins, the fusions containing additional binding domains were made to the N- or C-terminus of an VEGF monovalent antibody (VEGF MoAb), which is defined by a VEGF MoAb heavy chain 1 (hole) (SEQ ID NO: l) and a VEGF MoAb heavy chain 2 (knob) (SEQ ID NO: 2). An overview on these constructs is shown in Table la. a) Based on this MoAb, the bispecific, bivalent antigen binding protein of the invention BiMoMAb-N-Fab is made by fusing the Ang2i-LC06-CL-VL (SEQ ID NO: 4) via a (G4S)4 connector to the VEGF MoAb heavy chain 1 (hole) (SEQ ID NO: l) and the Ang2i-LC06-CHl-VH (SEQ ID NO: 3) domain via a (G4S)4 connector to the VEGF MoAb heavy chain 2 (knob) (SEQ ID NO: 2). b) Based on this VEGF MoAb, the bispecific, bivalent antigen binding protein of the invention BiMoMAb-N-Fv is made by fusing the Ang2i-Vl (SEQ ID NO: 6) via a (G4S)4 connector to the N-terminus of HC1 (SEQ ID NO: l)of the VEGF MoAb and the Ang2i-LC06-Vh (SEQ ID NO: 5) domain via a (G4S)4 connector to the HC2 (SEQ ID NO: 2)of the VEGF MoAb. c) Based on this VEGF MoAb, the bispecific, bivalent antigen binding protein of the invention BiMoMAb-C-scFab is made by fusing the Ang2i-LC06 scFab (SEQ ID NO: 7) via a (G4S)4 connector to the C-terminus of HC2 (SEQ ID NO: 2) of the VEGF MoAb and the VEGF-MoAb HC1 (SEQ ID NO: 1 remains unchanged (the SEQ ID NO: 1 is identical to SEQ ID NO: 12, see Sequence listing). Table la: Different bispecific, bivalent antigen binding proteins of the invention comrising as first antigen binding site under A) i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; and as second antigen binding site under B) different antigen binding peptides
(An "-" in the table means "not present")
Figure imgf000051_0001
Analogously (with an exchange of CHI and CL domain only in the monovalent first binding site) the following bispecific antigen binding proteins were generated. Table lb: Different bispecific, bivalent antigen binding proteins of the invention comprising as first antigen binding site under A) ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody; and as second antigen binding site under B) different antigen binding peptides
Figure imgf000052_0001
Example 1: Expression & purification of bispecific monovalent antibodies
<VEGF-ANG-2> BiMoMAh 1, BiMoMAb 2, BiMoMAb 3 and BiMoMAb 4
Heavy chain 1 and heavy chain 2 of the corresponding bispecific monovalent antibodies BiMoMAb 1, BiMoMAb 2 and BiMoMAb 3 and BiMOMab 4 were constructed in genomic expression vectors as described above). The plasmids were amplified in E. coli, purified, and subsequently transfected for transient expression of recombinant proteins in HEK293-F cells (utilizing Invitrogen's FreeStyle 293 system). After 7 days, HEK 293-F cell supernatants were harvested, filtered and the bispecific antibodies were purified by protein A and size exclusion chromatography. Homogeneity of all bispecific antibody constructs was confirmed by SDS-PAGE under non reducing and reducing conditions and analytical size exclusion chromatography. The expression yields were summarized in Table 2.
Table 2 summarizes the purifications of different BiMoMAb constructs.
Figure imgf000053_0001
The preparative SEC shows two prominent HMW peaks and one monomer peak for the BiMoMAb-N-Fab. The BiMoMAb-C-scFab shows two small HMW peaks and one well defined monomer peak. Chip based CE-SDS analysis shows for the BiMoMAb-N-Fab one band under reducing conditions with an apparent molecular weight of 97 kDa and 160 kDa under non reducing conditions. SDS-PAGE analysis of the BiMoMAb-C-scFab under reducing conditions shows two polypeptide heavy chains one carrying the C-terminal scFab fusion with a apparent molecular weight of 100 kDa and the other heavy chain without a fusion partner with an apparent molecular weight of 50 kDa. Under non reducing conditions the apparent molecular weight is about 160 kDa. MS experiments confirmed the integrity of the different BiMoMAb constructs. ESI-MS measurements after deglycosylation showed the expected molecular weight and confirmed the correct pairing of HC 1 and HC 2 in the purified monomer peak under non reducing conditions. Under reducing conditions and after deglycosylation the expected masses for all 4 constructs were obtained. The data is summarized in Table 3. Table 3: Summary of ESI-MS measurements. Antibodies were analyzed after deglycosylation with N-glycosidase F under reducing and non reducing conditions. Depicted are the average masses.
Figure imgf000054_0001
Example 2
Binding and Simultaneous binding of bispecific monovalent antibodies <VEGF-ANG-2> BiMoMAb-N-Fab, BiMoMAb-N-Fv and BiMoMAb-C-scFab
The binding of the Fab, scFab and Fv modules in the fusion domain and of the Fvs retained in the MoAb-module of the different bispecific antibody formats were compared to the binding of the 'wildtype' IgGs and different bispecific/tetravalent antibodies from which the binding modules and bispecific antibodies were derived. Two different assays were applied for these measurements:
1. hAng2 as analyte in solution: In this assay the bispecific antibody is
immobilized via an anti human Fc capturing antibody and hAng2 is in solution.
2. bispecific antibody as analyte in solution: In this assay the bispecific
antibody is in solution and the hAng2 is immobilized on the chip surface.
3. VEGF binding was tested using the assay 1 applying VEGF instead of
hAng2 in solution.
These analyses were carried out at equimolar concentrations by performing biochemical binding ELISAs and by applying Surface Plasmon Resonance (Biacore). For <VEGF-ANG-2> BiMoMAb-scFab and the BiMoMAb-N-Fab the VEGF binding shows a very high affinity with an apparent Kd of <0.1nM. Even though the affinity seems not to be altered there are differences in the association rate Ka for these 2 constructs. The association rate for the BiMoMAb-N-Fab seems to be slower than the association rate for the BiMoMAb-C-scFab (4E4 versus 1E5 M~V l) and for the the VEGF binding affinity is about that is described above that it binds to VEGF comparable to its parent antibody.
Table 4: Biacore of different multispecific antigen binding proteins of the invention to ANG2
Figure imgf000055_0001
Table 5: Biacore binding of different multispecific antigen binding proteins of the invention to VEGF
Biacore VEGF
Antibody Name ka (1/Ms) kd (1/s) KD (nM) MW [kDa]
BimoMab-C-scFab 1.01E+05 < 1E-06 < lE-10 150.00
(BiMoMAb3)
BimoMab-N-Fab 3.99E+04 < 1E-06 < lE-10 150.00
(BiMoMAbl) Example 3
Tie2 phosphorylation
In order to confirm that the anti-ANGPT2 related activities were retained in the bispecific monovalent BiMoMab <VEGF-ANGPT2> antibodies (N-Fab, C-scFab, N-Fv) it was shown that the BiMoMAbs interfere with ANGPT2 stimulated Tie2 phosphorylation in a comparable manner and that their binding properties in a monovalent manner were maintained compared to Fabs or scFvs.
In a first experiment the bispecific antibodies BiMoMAb-N-Fab and BiMoMAb-C- scFab showed a dose-dependent interference with ANGPT2 stimulated Tie2 phosphorylation. The IC50 values were higher than the ones obtained for bivalent binding molecules like the originating IgG (Ang2i-LC06) but comparable to those of the Fabs from Ang2i-LC06. The BiMoMAb-N-Fab interfered with ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx. 3320 ng/ml, BiMoMAb-C-scFab interfered with ANGPT2 stimulated Tie2 phosphorylation with a IC50 value of approx. 1504 ng/ml.
Taken together these data show that the bispecific monovalent <VEGF-ANGPT2> antibodies BiMoMAb-N-Fab and BiMoMAb-C-scFab interfere with ANGPT2 stimulated Tie2 phosphorylation in a dose dependent manner. The IC50 values are higher for monovalent binders, then for bivalent binder, but in a typical range for monovalent (Fab) binding and comparable to their mother clones Ang2i-LC06 as a Fab within the error of this cellular assay.
Table 6: Summary of the P-Tie2 ELISA for different BiMoMAb antibodies and corresponding references
Antibody Name IC50 [ng/ml] IC50 [nM] MW [kDa]
BiMoMAb-N-Fv n.d. n.d.
(BiMoMAb2)
BimoMab-C-scFab 1500 10 150 (BiMoMAb3)
BimoMab-N-Fab 3320 22 150 (BiMoMAb 1) Example 4
Inhibition of huANG-2 binding to Tie-2 (ELISA)
The interaction ELISA is performed on 384 well microtiter plates (MicroCoat, DE, Cat.No. 464718) at RT. After each incubation step plates are washed 3 times with PBST. ELISA plates were coated with 0.5 μg/ml Tie-2 protein (R&D Systems,
UK, Cat.No.313-TI) for at least 2 hours (h). Thereafter the wells are blocked with PBS supplemented with 0.2 % Tween-20 and 2 % BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutions of purified antibodies in PBS are incubated together with 0.2 μ^πιΐ huAngiopoietin-2 (R&D Systems, UK, Cat.No. 623-AN) for 1 h at RT. After washing a mixture of 0.5 μg/ml biotinylated anti-Angiopoietin-2 clone
BAM0981 (R&D Systems, UK) and 1 :3000 diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat.No.l 1089153001) is added for 1 h. Thereafter the plates are washed 6 times with PBST. Plates are developed with freshly prepared ABTS reagent (Roche Diagnostics GmbH, DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT. Absorbance was measured at 405 nm.

Claims

Patent Claims
A multispecific antigen binding protein, comprising
A) a monovalent antigen binding protein, which specifically binds to a first antigen comprising i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody; or ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody;
B) an antigen binding peptide which specifically binds to a second antigen, wherein the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus or N-terminus of one or both heavy chains under A).
The multispecific antigen binding protein according to claim 1 characterized in being a bispecific, bivalent antibody.
The multispecific antigen binding protein according to any one of claims 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises i) a) a first heavy chain of an full length antibody; and b) a second modified heavy chain of said full length antibody, wherein the VH domain is replaced by the VL domain of said antibody and the CHI domain is replaced by the CL domain of said antibody.
The multispecific antigen binding protein according to any one of claims 1 to 2 characterized in that the monovalent antigen binding protein under A), comprises ii) a) a first modified heavy chain of an full length antibody, wherein the VH domain is replaced by the VL domain of said antibody; and b) a second modified heavy chain of said full length antibody, wherein the CHI domain is replaced by the CL domain of said antibody.
The multispecific antigen binding protein according to any one of claims 1 to 4 characterized in that the antigen binding peptide is selected from the group of a Fv fragment, Fab fragment, a scFv fragment, and a scFab fragment.
The multispecific antigen binding protein according to any one of claim 5 characterized in that the antigen binding peptide is a Fv fragment.
The multispecific antigen binding protein according to any one of claim 5 characterized in that the antigen binding peptide is a Fab fragment.
The multispecific antigen binding protein according to any one of claim 5 characterized in that the antigen binding peptide is a scFv fragment.
The multispecific antigen binding protein according to any one of claim 5 characterized in that the antigen binding peptide is a scFab fragment.
The multispecific antigen binding protein according to any one of claims 1 to 9 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the N-terminus of one or both heavy chains under A).
The multispecific antigen binding protein according to any one of claims 1 to 9 characterized in that the the antigen binding peptide under B) is fused via one or two peptide connectors to the C-terminus of one or both heavy chains under A).
12. The multispecific antigen binding protein according to claim 1 characterized in comprising a) the amino acid sequence of SEQ ID NO: 8; and b) the amino acid sequence of SEQ ID NO:9; or a) the amino acid sequence of SEQ ID NO: 10; and b) the amino acid sequence of SEQ ID NO:l 1; or a) the amino acid sequence of SEQ ID NO: 12; and b) the amino acid sequence of SEQ ID NO : 13 ; or a) the amino acid sequence of SEQ ID NO: 14; and b) the amino acid sequence of SEQ ID NO: 15;.
13. A method for the preparation of a multispecific antigen binding protein according to the invention comprising the steps of a) transforming a host cell with vectors comprising nucleic acid molecules encoding a multispecific antigen binding protein according to claims 1 to 12 b) culturing the host cell under conditions that allow synthesis of said multispecific antigen binding protein molecule; and c) recovering said multispecific antigen binding protein molecule from said culture.
14. Nucleic acid encoding the multispecific antigen binding protein claims 1 to 12. Vectors comprising nucleic acid according to claim 14 encoding multispecific antigen binding protein claims 1 to 12.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017055443A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-pd1 antibodies and methods of use
WO2018091580A1 (en) 2016-11-18 2018-05-24 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
WO2019202040A1 (en) 2018-04-18 2019-10-24 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
WO2022247030A1 (en) 2021-05-27 2022-12-01 江苏荃信生物医药股份有限公司 ANTI-HUMAN INTERFERON α RECEPTOR 1 MONOCLONAL ANTIBODY AND APPLICATION THEREOF
WO2023029281A1 (en) 2021-09-03 2023-03-09 江苏荃信生物医药股份有限公司 Anti-human tslp monoclonal antibody and use thereof
WO2023029280A1 (en) 2021-09-03 2023-03-09 江苏荃信生物医药股份有限公司 Anti-human interleukin-33 monoclonal antibody and use thereof
JP2023518400A (en) * 2020-03-17 2023-05-01 システィミューン， インク． Micro-guidance and navigation control (miniGNC) antibody-like proteins, methods of making and using the same
WO2024038183A1 (en) * 2022-08-18 2024-02-22 Immunocore Limited Multi-domain binding molecules

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027011A1 (en) * 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
US20040033561A1 (en) * 2001-10-19 2004-02-19 Millennium Pharmaceuticals, Inc. Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor
WO2009080254A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027011A1 (en) * 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
US20040033561A1 (en) * 2001-10-19 2004-02-19 Millennium Pharmaceuticals, Inc. Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor
WO2009080254A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017055443A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-pd1 antibodies and methods of use
WO2018091580A1 (en) 2016-11-18 2018-05-24 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
EP4335874A2 (en) 2016-11-18 2024-03-13 F. Hoffmann-La Roche AG Anti-hla-g antibodies and use thereof
US11981737B2 (en) 2016-11-18 2024-05-14 Hoffmann-La Roche Inc. Anti-HLA-G antibodies and use thereof
WO2019202040A1 (en) 2018-04-18 2019-10-24 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
JP2023518400A (en) * 2020-03-17 2023-05-01 システィミューン， インク． Micro-guidance and navigation control (miniGNC) antibody-like proteins, methods of making and using the same
JP7503642B2 (en) 2020-03-17 2024-06-20 システィミューン， インク． Miniature Guidance and Navigation Control (miniGNC) Antibody-Like Proteins, Methods of Making and Using The Same - Patent application
EP4121461A4 (en) * 2020-03-17 2024-08-07 Systimmune Inc Miniature guidance and navigation control (minignc) antibody-like proteins and methods of making and using thereof
WO2022247030A1 (en) 2021-05-27 2022-12-01 江苏荃信生物医药股份有限公司 ANTI-HUMAN INTERFERON α RECEPTOR 1 MONOCLONAL ANTIBODY AND APPLICATION THEREOF
WO2023029281A1 (en) 2021-09-03 2023-03-09 江苏荃信生物医药股份有限公司 Anti-human tslp monoclonal antibody and use thereof
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WO2024038183A1 (en) * 2022-08-18 2024-02-22 Immunocore Limited Multi-domain binding molecules

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