Single-Chain Multivalent Binding Proteins with Effector Function

Abstract

Multivalent binding peptides, including bi-specific binding peptides, having immunoglobulin effector function are provided, along with encoding nucleic acids, vectors and host cells as well as methods for making such peptides and methods for using such peptides to treat or prevent a variety of diseases, disorders or conditions, as well as to ameliorate at least one symptom associated with such a disease, disorder or condition.

1 . A multivalent single-chain binding protein with effector function comprising
 a. a first binding domain derived from an immunoglobulin or immunoglobulin-like molecule;   b. a constant sub-region providing an effector function, the immunoglobulin constant sub-region located C-terminal to the first binding domain;   c. a scorpion linker located C-terminal to the constant sub-region; and   d. a second binding domain derived from an immunoglobulin or immunoglobulin-like molecule located C-terminal to the constant sub-region;   thereby localizing the constant sub-region between the first binding domain and the second binding domain.   
     
     
         2 . The protein according to  claim 1  wherein at least one of the first binding domain and the second binding domain comprises a variable light region derived from a first immunoglobulin and a variable heavy region derived from a second immunoglobulin. 
     
     
         3 . The protein according to  claim 1  wherein at least one of the first binding domain and the second binding domain comprises a variable light region and a variable heavy region derived from the same immunoglobulin. 
     
     
         4 . The protein according to  claim 1  wherein the first binding domain and the second binding domain recognize the same molecular target. 
     
     
         5 . The protein according to  claim 1  wherein the first binding domain and the second binding domain recognize the same epitope. 
     
     
         6 . The protein according to  claim 1  wherein the first binding domain and the second binding domain recognize different molecular targets on the same eukaryotic cell, prokaryotic cell, virus, carrier, or object. 
     
     
         7 . The protein according to  claim 1  wherein the first binding domain and the second binding domain recognize molecular targets associated with physically distinct eukaryotic cells, prokaryotic cells, viruses, carriers or objects. 
     
     
         8 . The protein according to  claim 1  wherein at least one of the first binding domain and the second binding domain recognize at least one molecular target not associated with a eukaryotic cell, prokaryotic cell, virus, carrier or object. 
     
     
         9 . The protein according to  claim 1  wherein the first binding domain, the second binding domain and the constant sub-region are each derived from a human immunoglobulin. 
     
     
         10 . The protein according to  claim 1  wherein at least one of the first binding domain and the second binding domain recognizes a target on a cancer cell. 
     
     
         11 . The protein according to  claim 9  wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a Dendritic cell target, an NK cell target, a monocyte/macrophage cell target and an angiogenesis target. 
     
     
         12 . The protein according to  claim 10  wherein the cancer cell is a transformed hematopoietic cell. 
     
     
         13 . The protein according to  claim 12  wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of a B-cell target, a T-cell target, a dendritic cell target, a myeloid cell target, or an NK-cell target. 
     
     
         14 . The protein according to  claim 12  wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of CD5, CD10, CD11c, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD30, CD38, CD45, CD70, CD72, CD79a, CD79b, CD80, CD86, CD103, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8, a MHC Class II peptide, and B-cell antigen receptor. 
     
     
         15 . The protein according to  claim 1  comprising a sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 103, 105, 107, 109, 333, and 334. 
     
     
         16 . The protein according to  claim 1  wherein the constant sub-region recognizes an effector cell F C  receptor. 
     
     
         17 . The protein according to  claim 1  wherein the constant sub-region recognizes an effector cell surface protein selected from the group consisting of CD16, CD32a, CD32b, CD64, CD89, FcεR1, FcRn, and pIgR. 
     
     
         18 . The protein according to  claim 1  wherein the constant sub-region comprises a C H2  domain and a C H3  domain. 
     
     
         19 . The protein according to  claim 18 , wherein the C H3  domain is truncated and comprises a C-terminal sequence selected from the group consisting of SEQ ID NOS: 366, 367, 368, 369, 370 and 371. 
     
     
         20 . The protein according to  claim 1 , wherein the scorpion linker is derived from an interdomain region of an immunoglobulin superfamily member. 
     
     
         21 . The protein according to  claim 1 , wherein the scorpion linker is derived from an an immunoglobulin hinge. 
     
     
         22 . The protein according to  claim 21 , wherein the scorpion linker has a reduced, but non-zero, number of cysteines relative to the hinge from which the scorpion linker is derived, and wherein one cysteine corresponds to the N-terminal hinge cysteine of the immunoglobulin hinge. 
     
     
         23 . The protein according to  claim 22  wherein the scorpion linker is derived from an IgG1 hinge region and wherein the linker comprises 1 or 2 cysteines, further wherein the linker retains the cysteine corresponding to an N-terminal hinge cysteine of the IgG1 hinge region. 
     
     
         24 . The protein according to  claim 1 , wherein the scorpion linker is derived from the stalk region of a C-type lectin. 
     
     
         25 . The protein according to  claim 1 , wherein the scorpion linker comprises a sequence selected from the group consisting of SEQ ID NOS:373, 374, 375, 376, 377, 380 and 381. 
     
     
         26 . The protein according to  claim 1  further comprising an N-terminal proximal linker of at least about 5 amino acids attached to the constant sub-region and attached to the first binding domain, thereby localizing the linker between the constant sub-region and the first binding domain. 
     
     
         27 . The protein according to  claim 1  in which the binding affinity of at least one of the first binding domain and the second binding domain is about 10 −6 M to 10 −9  M. 
     
     
         28 . The protein according to  claim 1 , wherein the effector function is selected from the group consisting of antibody-dependent cell-mediated cytotoxicity (ADCC) complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) and F C R binding. 
     
     
         29 . The protein according to  claim 1 , wherein the in vivo half-life of the protein is at least 28 hours in a human. 
     
     
         30 . A pharmaceutical composition comprising the protein according to  claim 1  and a pharmaceutically acceptable adjuvant, carrier or excipient. 
     
     
         31 . A nucleic acid encoding a protein according to  claim 1 . 
     
     
         32 . A vector comprising a nucleic acid according to  claim 31 . 
     
     
         33 . A host cell comprising a vector according to  claim 32 . 
     
     
         34 . A method of producing the protein according to  claim 1  comprising:
 a. introducing a nucleic acid encoding the protein according to  claim 1  into a host cell; and   b. incubating the host cell under conditions suitable for expression of the protein, thereby expressing the protein.   
     
     
         35 . The method according to  claim 34  further comprising isolating the protein. 
     
     
         36 . A method of producing a nucleic acid encoding the protein according to  claim 1  comprising:
 a. covalently linking the 3′ end of a polynucleotide encoding a first binding domain derived from an immunoglobulin variable region to the 5′ end of a polynucleotide encoding a constant sub-region;   b. covalently linking the 5′ end of a polynucleotide encoding a scorpion linker to the 3′ end of the polynucleotide encoding the constant sub-region; and   c. covalently linking the 5′ end of a polynucleotide encoding a second binding domain derived from an immunoglobulin variable region to the 3′ end of the polynucleotide encoding the scorpion linker,   thereby generating a nucleic acid encoding a multivalent binding protein with effector function.   
     
     
         37 . The method according to  claim 36  wherein the polynucleotide encoding a first binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1 (anti-CD20 variable region), SEQ ID NO:3 (anti-CD28 variable region, oriented V L -V H ) and SEQ ID NO:5 (anti-CD28 variable region, oriented V H -V L ). 
     
     
         38 . The method according to  claim 36 , further comprising a linker polynucleotide inserted between the polynucleotide encoding a first binding domain and the polynucleotide encoding a constant sub-region, the linker polynucleotide encoding a peptide linker of at least 5 amino acids. 
     
     
         39 . The method according to  claim 36 , further comprising a linker polynucleotide inserted between the polynucleotide encoding a constant sub-region and the polynucleotide encoding a second binding domain, the linker polynucleotide encoding a peptide linker of at least 5 amino acids. 
     
     
         40 . A method of inducing damage to a target cell comprising contacting a target cell with a therapeutically effective amount of a protein according to  claim 1 . 
     
     
         41 . The method according to  claim 40  wherein the target cell is contacted in vivo by administration of the protein to an organism in need. 
     
     
         42 . A method of treating a cell proliferation disorder comprising administering a therapeutically effective amount of a protein according to  claim 1  to an organism in need. 
     
     
         43 . A method of treating a disorder selected from the group consisting of a cancer, an autoimmune disorder, an infectious disease and inflammation. 
     
     
         44 . The method according to  claim 43  wherein the protein is administered by in vivo expression of a nucleic acid encoding the protein according to  claim 1 . 
     
     
         45 . The method according to  claim 43  wherein the protein is administered by a route selected from the group consisting of intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection and direct tissue injection. 
     
     
         46 . A method of ameliorating a symptom associated with a disorder selected from the group consisting of a cancer, an autoimmune disorder, an infectious disease and inflammation comprising administering a therapeutically effective amount of a protein according to  claim 1  to an organism in need. 
     
     
         47 . The method according to  claim 46 , wherein the symptom is selected from the group consisting of pain, heat, swelling and joint stiffness. 
     
     
         48 . A method of treating an infection associated with an infectious agent comprising administering a therapeutically effective amount of the protein according to  claim 1  to a patient in need, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. 
     
     
         49 . A method of ameliorating a symptom of an infection associated with an infectious agent comprising administering an effective amount of the protein according to  claim 1  to a patient in need, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. 
     
     
         50 . A method of reducing the risk of infection attributable to an infectious agent comprising administering a prophylactically effective amount of the protein according to  claim 1  to a patient at risk of developing the infection, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. 
     
     
         51 . A kit comprising the protein according to  claim 1  and a set of instructions for administering the protein to treat a cell proliferation disorder or to ameliorate a symptom of the cell proliferation disorder. 
     
     
         52 . The multivalent single-chain binding protein according to  claim 1  wherein at least one of the first binding domain and the second binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. 
     
     
         53 . The protein according to  claim 52  wherein one of the first binding domain and the second binding domain specifically binds CD20. 
     
     
         54 . The protein according to  claim 53  wherein the other binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. 
     
     
         55 . The protein according to  claim 52  wherein one of the first binding domain and the second binding domain specifically binds CD79b. 
     
     
         56 . The protein according to  claim 55  wherein the other binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. 
     
     
         57 . The protein according to  claim 52  wherein one of the first binding domain and the second binding domain specifically binds a major histocompatibility complex class II peptide. 
     
     
         58 . The protein according to  claim 57  wherein the other binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. 
     
     
         59 . The protein according to  claim 52  wherein one of the first binding domain and the second binding domain specifically binds CD22. 
     
     
         60 . The protein according to  claim 59  wherein the other binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a major histocompatibility complex class II peptide. 
     
     
         61 . The protein according to  claim 52  wherein the protein has at least an additive effect on a target cell behavior relative to the sum of the effects of each of the binding domains, and wherein the protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD40, CD20-CD70, CD20-CD79a, CD20-CD79b, CD20-CD80, CD20-CD81, CD20-CD86, CD79b-CD19, CD79b-CD23, CD79b-CD30, CD79b-CD37, CD79b-CD72, CD79b-CD81, CD79b-major histocompatibility complex class II (CL II), CL II-CD19, CL II-CD30, CL II-CD37, CL II-CD72, CL II-CD79a, CL II-CD81, CD19-CD37, CD19-CD79a, CD37-CD79a, CD81-CD37, and CD81-CD72. 
     
     
         62 . The protein according to  claim 52  wherein the protein has a synergistic effect on a target cell behavior relative to the sum of the effects of each of the binding domains, and wherein the protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD79a, CD20-CD79b, CD20-CD81, CD79b-CD37, major histocompatibility complex class II (CL II)-CD19, CL II-CD30, CL II-CD37, and CL II-CD72. 
     
     
         63 . The protein according to  claim 52  wherein the protein has an inhibitory effect on a target cell behavior relative to the sum of the effects of each of the binding domains, and wherein the protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD22-CD19, CD22-CD21, CD22-CD23, CD22-CD30, CD22-CD37, CD22-CD40, CD22-CD70, CD22-CD72, CD22-CD79a, CD22-CD79b, and CD22-major histocompatibility complex class II (CL II). 
     
     
         64 . A method of identifying at least one of the binding domains of the multivalent binding molecule according to  claim 1  comprising:
 (a) linking an antibody specifically recognizing a first antigen and an antibody specifically recognizing a second antigen;   (b) contacting a target comprising at least one of the antigens with the composition of step (a); and   (c) measuring an activity of the target, wherein the activity is used to identify at least one of the binding domains of the multivalent binding molecule.   
     
     
         65 . The method according to  claim 64  wherein the target is a diseased cell. 
     
     
         66 . The method according to  claim 40  wherein the multivalent single-chain binding protein induces a synergistic amount of damage to the target cell compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. 
     
     
         67 . The method according to  claim 66  wherein the multivalent single-chain binding protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD40, CD20-CD79a, CD20-CD79b, CD20-CD81, CD79b-CD37, CD79b-CD81, major histocompatibility complex class II (CL II)-CD37, CL II-CD72 and CL II-CD79b. 
     
     
         68 . The method according to  claim 40  wherein the multivalent single-chain binding protein induces at least an additive amount of damage to the target cell compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. 
     
     
         69 . The method according to  claim 68  wherein the multivalent single-chain binding protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD40, CD20-CD70, CD20-CD79a, CD20-CD79b, CD20-CD80, CD20-CD81, CD20-CD86, CD79b-CD19, CD79b-CD23, CD79b-CD30, CD79b-CD37, CD79b-CD72, CD79b-CD81, CD79b-major histocompatibility complex class II (CL II), CL II-CD19, CL II-CD30, CL II-CD37, CL II-CD72, CL II-CD79a, CL II-CD81, CD19-CD37, CD19-CD79a, CD37-CD79a, CD81-CD37, and CD81-CD72. 
     
     
         70 . The method according to  claim 40  wherein the multivalent single-chain binding protein induces an inhibited amount of damage to the target cell compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. 
     
     
         71 . The method according to  claim 70  wherein the multivalent single-chain binding protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD22-CD19, CD22-CD21, CD22-CD23, CD22-CD30, CD22-CD37, CD22-CD40, CD22-CD70, CD22-CD72, CD22-CD79a, CD22-CD79b, and CD22-major histocompatibility complex class II (CL II). 
     
     
         72 . The method according to  claim 40  further comprising a plurality of multivalent single-chain binding proteins. 
     
     
         73 . The method according to  claim 72  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein induce a synergistic amount of damage to the target cell. 
     
     
         74 . The method according to  claim 72  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein induce at least an additive amount of damage to the target cell. 
     
     
         75 . The method according to  claim 72  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein induce an inhibited amount of damage to the target cell. 
     
     
         76 . A composition comprising a plurality of multivalent single-chain binding proteins according to  claim 1 . 
     
     
         77 . The composition according to  claim 76  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein are capable of inducing a synergistic amount of damage to a target cell. 
     
     
         78 . The composition according to  claim 76  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein are capable of inducing an additive amount of damage to a target cell. 
     
     
         79 . The composition according to  claim 76  wherein a binding domain of a first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein are capable of inducing an inhibited amount of damage to a target cell. 
     
     
         80 . A pharmaceutical composition comprising the composition according to  claim 76  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         81 . A kit comprising the composition according to  claim 76  and a set of instructions for administering the composition to damage a target cell.