Single-chain multivalent binding proteins with effector function

Abstract

Variations on the structural themes for multivalent binding molecules with effector function, or scorpions, will be apparent to those of skill in the art upon review of the present disclosure, and such variant structures are within the scope of the invention.

1 . A multivalent binding protein comprising, from amino to carboxyl terminus:
 a) a first binding domain;   b) a constant sub-region comprising an immunoglobulin hinge region, C H2  domain and a C H3  domain;   c) a C-terminus linker peptide; and   d) a second binding domain,   
       wherein the binding protein is capable of cross-linking two or more targets. 
     
     
         2 . The multivalent binding protein of  claim 1 , 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. 
     
     
         3 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a tumor antigen. 
     
     
         4 . The multivalent binding protein of  claim 3 , wherein the tumor antigen is selected from the group consisting of SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B; KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN; CARCINOMA-ASSOCIATED MUCIN; POLYMORPHIC EPITHELIAL MUCIN; PEM; PEMT; EPISIALIN; TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN; EMA; H23AG; PEANUT-REACTIVE URINARY MUCIN; PUM; and BREAST CARCINOMA-ASSOCIATED ANTIGEN DF3), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN; DAM10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR-15, Serologically defined breast cancer antigen NY-BR-16, Chromogranin A, parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6. 
     
     
         5 . The multivalent binding protein of  claim 3 , wherein the tumor antigen is prostate-specific membrane antigen. 
     
     
         6 . The multivalent binding protein of  claim 3 , wherein the tumor antigen is a CTCL tumor antigen. 
     
     
         7 . The multivalent binding protein of  claim 6 , wherein the CTCL tumor antigen is selected from the group consisting of CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-9, CTCL tumor antigen se20-4, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1 and CTCL tumor antigen se89-1. 
     
     
         8 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a B cell target. 
     
     
         9 . The multivalent binding protein of  claim 8 , wherein the B cell target is selected from the group consisting of CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw150. 
     
     
         10 . The multivalent binding protein of  claim 9 , wherein the B cell target is CD37. 
     
     
         11 . The multivalent binding protein of  claim 9 , wherein the B cell target is CD86 or CD80. 
     
     
         12 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a T-cell target. 
     
     
         13 . The multivalent binding protein of  claim 11 , wherein the T-cell target is selected from the group consisting of 2B4/SLAMF4, IL-2 R alpha, 4-1BB/TNFRSF9, IL-2 R beta, ALCAM, B7-1/CD80, IL-4 R, B7-H3, BLAME/SLAMF8, BTLA, IL-6 R, CCR3, IL-7 R alpha, CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R alpha, CCR7, IL-10 R beta, CCR8, IL-12 R beta 1, CCR9, IL-12 R beta 2, CD2, IL-13 R alpha 1, IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, Integrin alpha 4/CD49d, CD5, Integrin alpha E/CD103, CD6, Integrin alpha M/CD11b, CD8, Integrin alpha X/CD11c, Integrin beta 2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, Leukotriene B4 R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common gamma Chain/IL-2 R gamma, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP beta 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fc gamma RIII/CD16, TIM-6, GITR/TNFRSF18, TNF R1/TNFRSF1A, Granulysin, TNF R11/TNFRSF1B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-gamma R1, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP, IL-1 RI and TSLP R. 
     
     
         14 . The multivalent binding protein of  claim 12 , wherein the T-cell target is CD3. 
     
     
         15 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a NK cell target. 
     
     
         16 . The multivalent binding protein of  claim 14 , wherein the NK cell target is selected from the group consisting of 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc epsilon RII, LMIR6/CD300LE, Fc gamma R1/CD64, MICA, Fc gamma RIIB/CD32b, MICB, Fc gamma RIIC/CD32c, MULT-1, Fc gamma RIIA/CD32a, Nectin-2/CD112, Fc gamma RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, Fc Receptor-like 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1 alpha, Rae-1 beta, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j, Rae-1 gamma, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3. 
     
     
         17 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a monocyte/macrophage cell target. 
     
     
         18 . The multivalent binding protein of  claim 17 , wherein the monocyte/macrophage cell target is selected from the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common beta Chain, Integrin alpha 4/CD49d, BLAME/SLAMF8, Integrin alpha X/CD11c, CCL6/C10, Integrin beta 2/CD18, CD155/PVR, Integrin beta 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3, Leukotriene B4 R1, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc gamma R1/CD64, Osteopontin, Fc gamma RIIB/CD32b, PD-L2, Fc gamma RIIC/CD32c, Siglec-3/CD33, Fc gamma RIIA/CD32a, SIGNR1/CD209, Fc gamma RIII/CD16, SLAM, GM-CSF R alpha, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-gamma R1, TLR4, IFN-gamma R2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF4, IL-10 R alpha, ALCAM, IL-10 R beta, Aminopeptidase N/ANPEP, ILT2/CD85j, Common beta Chain, ILT3/CD85k, C1q R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin alpha 4/CD49d, CCR5, Integrin alpha M/CD11b, CCR8, Integrin alpha X/CD11c, CD155/PVR, Integrin beta 2/CD18, CD14, Integrin beta 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4 R1, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, Fc gamma R1/CD64, PSGL-1, Fc gamma RIII/CD16, RP105, G-CSF R, L-Selectin, GM-CSF R alpha, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6 R, TREM-2, CXCR1/IL-8 RA, TREM-3 and TREML1/TLT-1. 
     
     
         19 . The multivalent binding protein of  claim 1 , wherein at least one of the first binding domain and the second binding domain recognizes a Dendritic cell target. 
     
     
         20 . The multivalent binding protein of  claim 19 , wherein the Dendritic cell target is selected from the group consisting of CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB Ligand/TNFSF9, IL-12/IL-23 p40, 4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin alpha 4/CD49d, Aag, Integrin beta 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 R1, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, C1q R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2F-10/SLAMF9, Osteoactivin/GPNMB, Chem 23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc gamma R1/CD64, TLR3, Fc gamma RIIB/CD32b, TREM-1, Fc gamma RIIC/CD32c, TREM-2, Fc gamma RIIA/CD32a, TREM-3, Fc gamma RIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid R1. 
     
     
         21 . The multivalent binding protein of  claim 1 , wherein one of the first binding domain and the second binding domain binds a T cell and wherein the other of the first binding domain and the second binding domain binds a cancer cell. 
     
     
         22 . The multivalent binding protein of  claim 1 , wherein one of the first binding domain and the second binding domain binds a natural killer cell and wherein the other of the first binding domain and the second binding domain binds a cancer cell. 
     
     
         23 . The multivalent binding protein of  claim 1 , wherein one of the first binding domain and the second binding domain binds a T cell and wherein the other of the first binding domain and the second binding domain binds an infectious agent. 
     
     
         24 . The multivalent binding protein of  claim 1 , wherein one of the first binding domain and the second binding domain binds a natural killer cell and wherein the other of the first binding domain and the second binding domain binds an infectious agent. 
     
     
         25 . The multivalent binding protein of  claim 1 , wherein the protein directs cytotoxic T lymphocytes to infectious agents. 
     
     
         26 . The multivalent binding protein of  claim 1 , wherein the protein directs cytotoxic T lymphocytes to cancer cells. 
     
     
         27 . The multivalent binding protein of  claim 1 , wherein the protein directs natural killer cells to cancer cells. 
     
     
         28 . The multivalent binding protein of  claim 1 , wherein the protein directs natural killer cells to infectious agents. 
     
     
         29 . The multivalent binding protein of  claim 1 , wherein said binding protein does not comprise a C H1  domain. 
     
     
         30 . The multivalent binding protein of  claim 1 , wherein the hinge is a hinge selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinge and the hinge-like C H2  domain of IgM. 
     
     
         31 . The multivalent binding protein of  claim 30 , wherein the hinge is an IgG1 hinge. 
     
     
         32 . The multivalent binding protein of  claim 31 , wherein the hinge is a human IgG1 hinge with a mutation at one or two cysteine residues. 
     
     
         33 . The multivalent binding protein of  claim 1 , wherein the first binding domain and the second binding domain are about 20-30 Å apart to about 150-180 Å apart. 
     
     
         34 . The multivalent binding protein of  claim 1 , wherein the first binding domain and the second binding domain are about 20-30 Å apart to less than about 50 Å apart. 
     
     
         35 . The multivalent binding protein of  claim 1 , wherein the first binding domain and the second binding domain are about 50 Å apart. 
     
     
         36 . The multivalent binding protein of  claim 1 , wherein the binding protein exhibits a binding affinity of less than 10 −9  M or at least 10 −6  M for at least one of the first binding domain and the second binding domain. 
     
     
         37 . The multivalent binding protein of  claim 1 , wherein the C-terminus linker is derived from the interdomain region of an immunoglobulin superfamily member. 
     
     
         38 . The multivalent binding protein of  claim 37 , wherein the interdomain region of an immunoglobulin superfamily member comprises an interdomain region between the Ig V-like and Ig C-like regions selected from the group consisting of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166 and CD244. 
     
     
         39 . The multivalent binding protein of  claim 1 , wherein the C-terminus linker comprises an immunoglobulin core hinge region. 
     
     
         40 . The multivalent binding protein of  claim 1 , wherein the C-terminus linker is derived from a Type II Membrane Protein C-type lectin stalk region. 
     
     
         41 . The multivalent binding protein of  claim 39 , wherein the C-terminus linker is derived from the stalk region of a type II Membrane Protein C-type lectin selected from the group consisting of CD69, CD72, CD94, NKG2A, and NKG2D. 
     
     
         42 . The multivalent binding protein of  claim 1 , wherein the C-terminus linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 287, 289, 297, 305, 307, 309, 310, 311, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 346, 373, 374, 375, 376, 377, 380, and 381. 
     
     
         43 . The multivalent binding protein of  claim 1 , wherein the C-terminus linker does not comprise (Gly 4 Ser) 3 . 
     
     
         44 . The multivalent binding protein of  claim 1 , wherein the binding protein is a single chain molecule capable of forming a dimer by disulfide bond formation. 
     
     
         45 . The multivalent binding protein of  claim 1 , wherein the constant sub-region is capable of an effector function selected from the group consisting of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, relatively extended in vivo half-life (relative to the same molecule lacking a constant sub-region), FcR binding, and protein A binding. 
     
     
         46 . A nucleic acid encoding the protein of  claim 1 . 
     
     
         47 . A vector comprising the nucleic acid of  claim 46 . 
     
     
         48 . A host cell comprising the nucleic acid of  claim 46  or the vector of  claim 47 . 
     
     
         49 . A composition comprising the protein of  claim 1  and a pharmaceutically acceptable carrier.