Triple-specific binding protein, method, and use thereof

Abstract

To provide trispecific and / or trivalent binding proteins.SOLUTION: Disclosed herein is a trispecific and / or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, where a first pair of the polypeptides that form the binding protein possesses double binding domains having crossover tropism, and a second pair of the polypeptides has a single variable domain that forms a single antigen binding site. In some embodiments, the binding protein comprises a binding site that binds to CD28 polypeptide, a binding site that binds to CD3 polypeptide, and a binding site that binds to a third polypeptide that binds to, for example, a tumor targeting protein.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 831,572 filed on 9 April 2019 and EP Application No. EP19306311.2 filed on 8 October 2019, and the disclosures of these respective applications are incorporated herein by reference in their entirety.

[0002] Submission of sequence listings in ASCII text files. The following submission in ASCII text file is incorporated herein by reference in its entirety: a computer-readable format (CRF) sequence listing (filename: 183952032040SEQLIST.TXT, date recorded: April 6, 2020, size: 526KB).

[0003] field This disclosure relates to a triplicate and / or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein the first pair of polypeptides forming the binding protein has a bivariate domain having crossover orientation. This disclosure also relates to a method for producing a triplicate and / or trivalent binding protein and to the use of such a binding protein. [Background technology]

[0004] Monoclonal antibody-based biopharmaceuticals have become a crucial tool for novel drug development. Monoclonal antibody technology provides specific targeting, precise signaling, and / or payload delivery to specific cell populations, delivering long-lasting biological effects through its Fc function. Efforts in antibody engineering have enabled the development of bispecific antibodies, combining the specificity of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development. Newly discovered neutralizing antibodies with improved broadness and potency may offer more options for developing biopharmaceuticals to treat complex diseases, such as cancer, arthritis, and / or inflammatory disorders.

[0005] Immuno-oncology is a promising, emerging therapeutic approach for disease management in cancer. The immune system is the front line of defense against the onset and progression of cancer. There is now strong evidence that T cells can control tumor growth and prolong the survival of cancer patients at both the early and late stages of the disease. However, tumor-specific T cells are limited in numerous ways that prevent them from controlling the disease.

[0006] As part of human adaptive immunity, T-cell immunity plays a crucial role in controlling viral infections and cancer, eliminating infected and, in some cases, malignant cells, resulting in viral infection clearance or cancer cure. In chronic infectious diseases, such as herpesvirus infections (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans through various mechanisms, including immunosuppression, T-cell exhaustion, and latent establishment. Nevertheless, viral infection generally induces viral antigen-specific immunity, including antigen-specific CD8 T cells that can readily recognize infected cells for control or elimination through cytokine release or cytotoxic T-cell (CTL)-mediated killing processes.

[0007] Therefore, activation and / or amplification of viral antigen-specific T cells in vivo and / or ex vivo may provide a therapeutic strategy for chronic viral infections.

[0008] All references made herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference as a whole, in particular as each individual reference is incorporated by reference. [Overview of the project] [Problems that the invention aims to solve]

[0009] To meet these and other needs, triplicate binding proteins (e.g., antibodies) forming three antigen-binding sites are provided herein. These binding proteins can specifically bind to one, two, or three antigen targets or target proteins, e.g., CD28, CD3, and tumor target proteins. Some tumors express specific antigens. For example, amplification and overexpression of HER2 can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung, and prostate cancers. Optimal activation of T cells requires two factors: 1. antigen recognition and 2. co-stimulation. Using the triplicate binding proteins described herein, signal 1 is provided by the agonist anti-CD3 binding site, and signal 2 is provided by the agonist anti-CD28 binding site. The triplicate binding proteins recruit T cells to the tumor via binding sites that recognize HER2, CD38, or another tumor target protein, and activate the engaged T cells via anti-CD3 and CD28. The resulting activation induces the potential for immune cell-mediated killing of nearby tumor cells. Additionally, the anti-CD3 binding site is described along with high-affinity binding to the human CD3 polypeptide and the removal of potential manufacturing defects (e.g., deamide sites).

[0010] Further provided herein are anti-CD38 / CD28×CD3 trispecific antibodies developed and evaluated for their potential in T cell activation and subsequent proliferation and / or amplification of antigen-specific T cells. These trispecific antibodies can effectively expand and proliferate CD4 and CD8 effector and memory populations in vitro, including antigen-specific CD8 T central memory and effector memory cells. In particular, in vitro expansion and proliferation of CMV, EBV, HIV-1, and influenza-specific CD8 central memory and effector memory cells has been demonstrated. The anti-CD38 / CD28×CD3 trispecific antibodies described herein exhibit novel characteristics by engaging with CD3 / CD28 / CD38 and providing a signaling pathway for stimulating and expanding T cells, which may provide an effective strategy for treating chronic infectious diseases, such as HSV, CMV, EBV, HIV-1, and HBV infections.

[0011] To meet these and other needs, binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides) are provided herein, including monospecific, bispecific, or tripspecific binding proteins having at least one antigen-binding site that binds to the CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells near cancer cells, subsequently activate them, and promote the killing of activated T cells in adjacent cancer cells via the granzyme / perforin mechanism, providing a mode of action for antitumor activity distinct from anti-CD38 antibodies, e.g., DARZALEX® (daratumumab). Furthermore, the ability to bind to both human and cynomolgus monkey CD38 polypeptides allows for easy testing of binding proteins in preclinical toxicological studies, for example, to evaluate their safety profiles for later clinical use. [Means for solving the problem]

[0012] In some embodiments, a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain has the formula: V -L1-V L1 -L2-C L [I] and comprises the structure represented by: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -hinge-C H2 -C H3 [II] and comprises the structure represented by: The third polypeptide chain has the formula: V H3 -C H1 -hinge-C H2 -C H3 [III] and comprises the structure represented by: The fourth polypeptide chain has the formula: V L3 -C L [IV] and comprises the structure represented by: where: V L1 is the first immunoglobulin light chain variable domain; V L2 is the second immunoglobulin light chain variable domain; V L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; C L is the immunoglobulin light chain constant domain; C H1 is the immunoglobulin C H1 heavy chain constant domain; C H2is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 It forms the first antigen-binding site; V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain is QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, and comprises a CDR-L1 sequence containing the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and V H3 and V L3 It forms a third antigen-binding site. The aforementioned binding protein is provided herein.

[0013] In some embodiments, the first binding site binds to the CD28 polypeptide. In some embodiments, V H1The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and a CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51), V L1 The domain includes a CDR-L1 sequence containing the amino acid sequence of QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53), and a CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). In some embodiments, V H1 The domain is The amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) is included and / or V L1 The domain is Contains the amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92).

[0014] In some embodiments, V L2The CDR-L1 sequence of the domain includes an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61), and QSLVHENLRTY (SEQ ID NO: 62). In some embodiments, the binding protein of the Disclosure includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antigen-binding site comprising an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).In some embodiments, the binding protein of the present disclosure comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding protein of the Disclosure comprises an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, V. H2 The domain is The amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) contains and / or V L2 The domain is, DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSL The amino acid sequence is selected from the group consisting of IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, V H2 The domain is The amino acid sequence includes QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302), and / or V L2 The domain is, DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 96), DIVMTQTPLSLS The amino acid sequence is selected from the group consisting of VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the binding protein of this disclosure includes an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93 and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding protein of the Disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 302 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding protein of the Disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 96. In some embodiments, the binding protein of the Disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 97.In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site comprising an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93 and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 98.

[0015] In some embodiments, the third antigen-binding site binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (e.g., human CD38 polypeptide). 38 polypeptides). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., human HER2 polypeptide). In some embodiments, tumor target proteins of the Disclosure include A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 ( FCER2 (also known as an IgE receptor), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (B CD278 (also known as 7H3), CD279 (also known as ICOS), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1,EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LA Examples of antigen targets include, but are not limited to, G3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0016] In some embodiments, the third antigen-binding site binds to the human CD38 polypeptide. In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), V L3 The domain contains the CDR-L1 sequence, which includes the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), and the amino acid sequence of GAS (SEQ ID NO: 17). It includes a CDR-L2 sequence containing an acid sequence, and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence containing the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR-H2 sequence containing the amino acid sequence of INPGSGST (SEQ ID NO: 26), and a CDR-H3 sequence containing the amino acid sequence of ARYAYGY (SEQ ID NO: 27), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QNVGTA (SEQ ID NO: 28), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 29), and a CDR-L3 sequence containing the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence containing the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36).

[0017] In some embodiments, V H3The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence containing the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence containing the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). In some embodiments, V H3 The domain is The amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79) contains and / or V L3 The domain is The amino acid sequence includes DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80). In some embodiments, V H3 The domain is The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81) is included and / or V L3 The domain is EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQ It contains the amino acid sequence PEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82). In some embodiments, V H3 The domain is The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83) contains and / or V L3 The domain is It contains the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84). In some embodiments, V H3 The domain is The amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87) is included and / or V L3 The domain is The amino acid sequence DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88) is included. In some embodiments, V H3 The domain is The amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89) contains and / or V L3 The domain is The amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90) is included. In some embodiments, V H3 The domain is The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85) is included and / or V L3 The domain is Contains the amino acid sequence DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (Sequence ID 86).

[0018] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 159. In some embodiments, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence identical to that of SEQ ID NO: 160 by at least 95%; the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence identical to that of SEQ ID NO: 161 by at least 95%; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence identical to that of SEQ ID NO: 162 by at least 95%; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence identical to that of SEQ ID NO: 163 by at least 95%. In some embodiments, the first polypeptide chain includes SEQ ID NO: 164 The first polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 167. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 171. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence identical to that of SEQ ID NO: 172 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence identical to that of SEQ ID NO: 173 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence identical to that of SEQ ID NO: 174 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence identical to that of SEQ ID NO: 175 by at least 95%.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence identical to that of SEQ ID NO: 176 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence identical to that of SEQ ID NO: 177 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence identical to that of SEQ ID NO: 178 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence identical to that of SEQ ID NO: 179 by at least 95%. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 184. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence identical to that of SEQ ID NO: 185 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence identical to that of SEQ ID NO: 186 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence identical to that of SEQ ID NO: 187 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence identical to that of SEQ ID NO: 188 by at least 95%.

[0019] In some embodiments, the third antigen-binding site binds to the human HER2 polypeptide. In some embodiments, V H3The domain includes a CDR-H1 sequence containing the amino acid sequence GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8). L3 Domaine The sequence includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), V L3The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, V H3 The domain is, EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (Sequence ID 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGS The amino acid sequence includes GFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and / or V L3 The domain is, It includes the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78). In some embodiments, V H3 The V domain includes the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or the V L3 domain includes the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, V H3 The V domain includes the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and / or the V L3 domain includes the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, V H3 The V The amino acid sequence contains EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) and / or V L3 The domain is The amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) is included. In some embodiments, V H3 The domain is The amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74) is included and / or V L3 The domain is The amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) is included. In some embodiments, V H3 The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76) contains the amino acid sequence and / or V L3 The domain is It includes the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, V H3 The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or V L3 The domain is It includes the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78).

[0020] In some embodiments, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 103. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 107. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 115.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 123. In some embodiments, the first polypeptide chain... The first polypeptide chain contains at least 95% identical amino acids to the amino acid sequence of SEQ ID NO: 124 or the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain contains at least 95% identical amino acids to the amino acid sequence of SEQ ID NO: 125 or the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain contains at least 95% identical amino acids to the amino acid sequence of SEQ ID NO: 126 or the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 127. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 131. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence identical to that of SEQ ID NO: 132 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence identical to that of SEQ ID NO: 133 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence identical to that of SEQ ID NO: 134 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence identical to that of SEQ ID NO: 135 by at least 95%.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 139. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence identical to that of SEQ ID NO: 140 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence identical to that of SEQ ID NO: 141 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence identical to that of SEQ ID NO: 142 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence identical to that of SEQ ID NO: 143 by at least 95%. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 147.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 151. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 152. The first polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain contains at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 288 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 289 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 289. In some embodiments, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 290 or an amino acid sequence identical to that of SEQ ID NO: 290 by at least 95%; the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 291 or an amino acid sequence identical to that of SEQ ID NO: 291 by at least 95%; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 292 or an amino acid sequence identical to that of SEQ ID NO: 292 by at least 95%; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 293 or an amino acid sequence identical to that of SEQ ID NO: 293 by at least 95%.In some embodiments, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 294 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 294; the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 295 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 295; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 296 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 296; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 297. In some embodiments, the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 298 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 298; the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 299 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 299; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 300 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 300; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 301 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 301.

[0021] In some embodiments, which can be combined with any other embodiments described herein, at least one of L1, L2, L3, or L4 is independently 0 amino acid length. In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acid length or include a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1, L2, L3, and L4 are each independently GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1 includes the sequence GQPKAAP (sequence number 67), L2 includes the sequence TKGPS (sequence number 68), L3 includes the sequence S, and L4 includes the sequence RT. In some embodiments, at least one of L1, L2, L3, or L4 includes the sequence DKTHT (sequence number 66). In some embodiments, L1, L2, L3, and L4 are sequences Includes DKTHT (sequence number 66).

[0022] In some embodiments, which can be combined with any other embodiments described herein, the hinges of the second and third polypeptide chains are C H2 -C H3 The domain is human IgG4 hinge-C. H2 -C H3 It is a domain, and hinge-C H2 -C H3 Each domain contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C. H2 -C H3It is a domain, and hinge-C H2 -C H3 Each domain contains amino acid substitutions at positions corresponding to positions 233–236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and deletion at 236. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG4 hinge-C. H2 -C H3 It is a domain, and hinge-C H2 -C H3 Each domain contains amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG1 hinge-C. H2 -C H3 It is a domain, and hinge-C H2 -C H3 Each domain contains amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 The domain is human IgG1 hinge-C. H2 -C H3 It is a domain, and hinge-C H2 -C H3 Each domain contains amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S. In some embodiments, the hinge-C of the second polypeptide chain is H2 -C H3The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; hinge-C of the third polypeptide chain. H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. In some embodiments, the hinge-C of the second polypeptide chain is present. H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; and the hinge-C of the third polypeptide chain. H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

[0023] In some embodiments, isolated nucleic acid molecules comprising a nucleotide sequence encoding a binding protein of any one of the embodiments described above are provided herein. In some embodiments, an expression vector comprising a nucleic acid molecule of any one of the embodiments described above is provided herein. In some embodiments, an isolated host cell comprising a nucleic acid molecule of any one of the embodiments described above or an expression vector of any one of the embodiments described above is provided herein. In some embodiments, the host cell is a mammalian or insect cell.

[0024] In some embodiments, pharmaceutical compositions comprising any one of the above embodiments' binding proteins and a pharmaceutically acceptable carrier are provided herein.

[0025] In some embodiments, the method for preventing and / or treating cancer in a patient A method is provided herein that comprises administering to a patient a therapeutically effective amount of at least one of the above embodiments of a binding protein or pharmaceutical composition. In some embodiments, a binding protein or pharmaceutical composition according to one of the above embodiments for use in a method of preventing and / or treating cancer in a patient is provided herein, wherein the method comprises administering to a patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, a binding protein or pharmaceutical composition according to one of the above embodiments for use in the manufacture of a medicament for preventing and / or treating cancer in a patient is provided herein.

[0026] In some embodiments, at least one binding protein is administered in combination with a chemotherapeutic agent. In some embodiments, the patient is human.

[0027] In some embodiments, the third antigen-binding site binds to a human CD38 polypeptide, and cancer cells from an individual or patient express CD38. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, prior to administration of the binding protein, the patient is treated with daratumumab without a washout period.

[0028] In some embodiments, the third antigen-binding site binds to a human HER2 polypeptide, and cancer cells from an individual or patient express HER2. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

[0029] In some embodiments, a method for expanding and proliferating virus-specific memory T cells comprises contacting the virus-specific memory T cells with a binding protein, wherein the binding protein comprises four polypeptide chains forming three antigen-binding sites, and the first polypeptide chain is given the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain is QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, and includes a CDR-L1 sequence containing the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), V H3 and V L3 This forms a third antigen-binding site that binds to the CD38 polypeptide. The method described herein is provided.

[0030] In some embodiments, a binding protein for use in the expansion and proliferation of virus-specific memory T cells comprises four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain consists of a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55) and a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56). The sequence includes the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and V L2 The domain is QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, and includes a CDR-L1 sequence containing the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), V H3 and V L3 A binding protein for the use described herein is provided, which forms a third antigen-binding site that binds to the CD38 polypeptide.

[0031] In some embodiments, virus-specific memory T cells come into contact with binding proteins in vitro or ex vivo. In some embodiments, contact of virus-specific memory T cells with binding proteins induces activation and / or proliferation of the virus-specific memory T cells.

[0032] In some embodiments, a method for expanding and proliferating T cells comprises contacting T cells with a binding protein in vitro or ex vivo, wherein the binding protein comprises four polypeptide chains forming three antigen-binding sites, and the first polypeptide chain is of formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain is QSLVHX1NX2X3TY, where X The CDR-L1 sequence includes the amino acid sequence QSLVHX1NX2X3TY (SEQ ID NO: 180), where 1 is E or Q, X2 is A or L, and X3 is Q, R, or F; the CDR-L2 sequence includes the amino acid sequence KVS (SEQ ID NO: 64); and the CDR-L3 sequence includes the amino acid sequence GQGTQYPFT (SEQ ID NO: 65). H3 and VL3 This forms a third antigen-binding site that binds to the CD38 polypeptide. The method described herein is provided.

[0033] In some embodiments, a binding protein for use in a method for expanding and proliferating T cells, comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3is the third immunoglobulin heavy chain variable domain; C L is the immunoglobulin light chain constant domain; C H1 is the immunoglobulin C H1 heavy chain constant domain; C H2 is the immunoglobulin C H2 heavy chain constant domain; C H3 is the immunoglobulin C H3 heavy chain constant domain; The hinge is the immunoglobulin hinge region connecting the C H1 and C H2 domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a crossover light chain - heavy chain pair; V H1 and V L1 form the first antigen - binding site that binds to the CD28 polypeptide, and V H2 and V L2 form the second antigen - binding site that binds to the CD3 polypeptide, and V H2 domain comprises a CDR - H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR - H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR - H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and V L2 domain comprises a CDR - L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, X3 is Q, R, or F, of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR - L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR - L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and V H3 and V L3 form the third antigen - binding site that binds to the CD38 polypeptide, Binding proteins for such use are provided herein.

[0034] In some embodiments, the T cell is a memory T cell or an effector T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

[0035] In some embodiments, a method of treating chronic viral infection, comprising administering to an individual or patient in need thereof an effective amount of a binding protein, the binding protein comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] comprising the structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 -hinge-C H2 -C H3 [II] comprising the structure represented by The third polypeptide chain has the formula: V H3 -C H1 -hinge-C H2 -C H3 [III] comprising the structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] comprising the structure represented by where V L1 is the first immunoglobulin light chain variable domain; V L2 is the second immunoglobulin light chain variable domain; V L3 is the third immunoglobulin light chain variable domain; V H1This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain is QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, and includes a CDR-L1 sequence containing the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), VH3 and V L3 This forms a third antigen-binding site that binds to the CD38 polypeptide. The method described herein is provided.

[0036] In some embodiments, a binding protein for use in a method for treating chronic viral infections, comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; Polypeptides of formula I and formula II form a crossover light-heavy chain pair; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain is QSLVHX1NX2X3TY, where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, and includes a CDR-L1 sequence containing the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), V H3 and V L3 This forms a third antigen-binding site that binds to the CD38 polypeptide. The method includes administering to an individual or patient in need thereof an effective amount of a binding protein. Binding proteins for such use are provided herein.

[0037] In some embodiments, the individual or patient is human. In some embodiments, the binding protein is administered to the individual or patient in a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier. In some embodiments, administration of the binding protein results in activation and / or proliferation of virus-specific memory T cells in the individual or patient.

[0038] In some embodiments, which can be combined with any other embodiments described herein, the memory T cells are CD8+ or CD4+ memory T cells. In some embodiments, the memory T cells are central memory T cells (T CM ) or effector memory T cells (T EM ).

[0039] In some embodiments, which can be combined with any other embodiments described herein, the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-Barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2).

[0040] In some embodiments, which can be combined with any other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide.

[0041] In some embodiments, a vector system is provided herein that includes one or more vectors encoding first, second, third, and fourth polypeptide chains of any one of the above embodiments of a binding protein. In some embodiments, the vector system includes a first vector encoding a first polypeptide chain of the binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein.

[0042] In some embodiments, a kit is provided herein comprising one, two, three, or four polypeptide chains of binding proteins according to any one of the embodiments described above. In some embodiments, the kit further includes instructions for use of the polypeptide chains or binding proteins according to any of the methods or uses described herein, for example.

[0043] In some embodiments, a kit is provided herein comprising one, two, three, or four polynucleotides according to any one of the embodiments described above. In some embodiments, a polynucleotide kit comprising (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a polynucleotide of SEQ ID NO: 200 (d) A fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 201, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 202, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 203, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 204; (e) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 205, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 206, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 207, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 208; (f) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 209, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 210, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 211, and a polynucleotide of SEQ ID NO: 212 (g) A fourth polynucleotide containing the nucleotide sequence; (h) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 213, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 214, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 215, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 216; (i) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 221, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 218, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 219, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 220; (j) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 225, and a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 226 (k) a polynucleotide containing the polynucleotide sequence of SEQ ID NO: 227, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 228; (k) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 229, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 230, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 231, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 232; (l) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 233, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 234, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 235, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 236; (m) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 237, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 238, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 239, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 240;(n) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 241, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 242, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 243, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 244; (o) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 245, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 246, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 247, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 248; (p) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 249, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 250, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 251, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 252; (q) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 253, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 254, (r) A third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 255, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 256; (r) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 257, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 258, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 259, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 260; (s) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 261, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 262, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 263, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 264; (t) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 265, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 266, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 267, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 268;(u) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 269, the polynucleotide of SEQ ID NO: 270; A kit comprising one, two, three, or four polynucleotides is provided herein, comprising (v) a first polynucleotide comprising the polynucleotide sequence of sequence number 273, a second polynucleotide comprising the polynucleotide sequence of sequence number 274, a third polynucleotide comprising the polynucleotide sequence of sequence number 275, and a fourth polynucleotide comprising the polynucleotide sequence of sequence number 276.

[0044] It should be understood that one, some, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the subsequent detailed description. [Brief explanation of the drawing]

[0045] [Figure 1A] A schematic diagram of a triple-specific binding protein is provided, comprising four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and HER2. The first pair of polypeptides has a dual variable domain with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, while the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes HER2. The triple-specific binding protein shown in Figure 1A uses a constant region with a "knob-into-hole" mutation, in which case the knob is located in the second pair of polypeptides along with the single variable domain. [Figure 1B]Figure 1B provides the change multiplier (relative to the parent) in binding affinity for anti-CD28 / CD3 / HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. Mutations 3233QQ~QEQ (from top to bottom) refer to mutations introduced into residues 32~35 of the VL domain of the anti-CD3 binding site (indicated by *); the remaining mutations were introduced into the VH or VL domain (indicated by #; numbering follows Kabat) of the trastuzumab anti-HER2 binding site. In the case of mutations in the anti-HER2 binding site, mutation 30Q was introduced into the VL domain and the remaining mutations into the VH domain. Binding affinity was measured by ELISA, and the values ​​provided are relative to the parental trispecific antibody. [Figure 1C] Figure 1C shows the binding curves of the tripspecific antibodies that bind to human HER2, human CD28, and CD3 as determined by ELISA. [Figure 1D] Figure 1D illustrates the proposed mechanism of action of HER2 / CD28×CD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing. [Figure 2-1]Figure 2A provides a schematic diagram of a triple-specific binding protein containing four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and CD38. The first pair of polypeptides has a dual variable domain with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, while the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes CD38. The triple-specific binding protein shown in Figure 2A utilizes an IgG4 constant region with a "knob-into-hole" mutation, in which case the knob is located in the second pair of polypeptides along with the single variable domain. Figures 2B-2E show the binding affinity of the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA tripspecific antibody, which has the indicated anti-CD38 binding domain, to the target antigens human CD38 (Figure 2B), cynomolgus monkey CD38 (Figure 2C), human CD3 (Figure 2D), and human CD28 (Figure 2E), as measured by ELISA. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Continuation of Figure 2-2. [Figure 3] Figure 3 shows a competitive SPR assay for binding to CD38 by daratumumab and an anti-CD38 monospecific antibody, both possessing the indicated anti-CD38 binding domain. When the antibody recognized an epitope on CD38 different from that of daratumumab, injection of the antibody resulted in an increase in the SPR signal. When the antibody recognized an epitope that overlapped with that of daratumumab, injection of the antibody did not increase the SPR signal. [Figure 4]Figures 4A-4B show the in vitro cell-killing activity of the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA triplicate antibody, which possesses the indicated anti-CD38 binding domain, against human multiple myeloma NCI-H929 cells (CD38+ / CD28+). The assay was performed in the presence of either a 5 nM isotype control antibody (Figure 4A) or daratumumab (Figure 4B). In the presence of daratumumab, the triplicate antibody continued to exhibit cell-killing activity. [Figure 5] Figures 5A-5B show the in vitro cell-killing activity of the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody, which possesses the indicated anti-CD38 binding domain, against human lymphoma OCI-Ly19 cells (CD38+ / CD28-). The assay was performed in the presence of 5 nM isotype control antibody (Figure 5A) or daratumumab (Figure 5B). Daratumumab caused a decrease in the cell-killing activity of the anti-CD38 / CD28×CD3 trispecific antibody. [Figure 6-1]Figures 6A–6J characterize the in vitro T cell subset expansion and proliferation in PBMCs collected from CMV-infected donor D in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated surrogate anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify CMV-specific CD8+ T cells (Figures 6A–6B), CMV-specific Tcm CD8+ cells (Figures 6C–6D), and CMV-specific Tem CD8+ cells (Figures 6E–6F). In addition, the percentages of CMV-specific Tcm (Figure 6G-6H) and Tem (Figure 6I-6J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-responsive manner with different potencies and kinetics. [Figure 6-2] Continuation of Figure 6-1. [Figure 6-3] Continuation of Figure 6-2. [Figure 6-4] Continuation of Figure 6-3. [Figure 6-5] Continuation of Figure 6-4. [Figure 7-1]Figures 7A–7J characterize the in vitro T cell subset expansion and proliferation in PBMCs collected from CMV-infected donor E in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody possessing the indicated anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). Antibodies shown from top to bottom in the legend are shown from left to right in the graph. T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM. Flow cytometry was used to quantify CMV-specific CD8+ T cells (Figures 7A-7B), CMV-specific Tcm CD8+ cells (Figures 7C-7D), and CMV-specific Tem CD8+ cells (Figures 7E-7F). In addition, the percentages of CMV-specific Tcm (Figures 7G-7H) and Tem (Figures 7I-7J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 7-2] Continuation of Figure 7-1. [Figure 7-3] Continuation of Figure 7-2. [Figure 7-4] Continuation of Figure 7-3. [Figure 7-5] Continuation of Figure 7-4. [Figure 8-1]Figures 8A–8J characterize the in vitro T cell subset expansion and proliferation in PBMCs collected from EBV-infected donor C in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated substituted anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 8A–8B), CMV-specific Tcm CD8+ cells (Figures 8C–8D), and CMV-specific Tem CD8+ cells (Figures 8E–8F). In addition, the percentages of EBV-specific Tcm (Figure 8G-8H) and Tem (Figure 8I-8J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 8-2] Continuation of Figure 8-1. [Figure 8-3] Continuation of Figure 8-2. [Figure 8-4] Continuation of Figure 8-3. [Figure 8-5] Continuation of Figure 8-4. [Figure 9-1]Figures 9A–9B illustrate the characterization of in vitro T cell subset expansion and proliferation in PBMCs collected from EBV-infected donor D in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated substituted anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A–9B), EBV-specific Tcm CD8+ cells (Figures 9C–9D), and EBV-specific Tem CD8+ cells (Figures 9E–9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 9-2] Continuation of Figure 9-1. [Figure 9-3] Continuation of Figure 9-2. [Figure 9-4] Continuation of Figure 9-3. [Figure 10]Figures 9A–9B illustrate the characterization of in vitro T cell subset expansion and proliferation in PBMCs collected from EBV-infected donor D in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated substituted anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A–9B), EBV-specific Tcm CD8+ cells (Figures 9C–9D), and EBV-specific Tem CD8+ cells (Figures 9E–9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 11]Figures 9A–9B illustrate the characterization of in vitro T cell subset expansion and proliferation in PBMCs collected from EBV-infected donor D in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated substituted anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A–9B), EBV-specific Tcm CD8+ cells (Figures 9C–9D), and EBV-specific Tem CD8+ cells (Figures 9E–9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 12]Figures 9A–9B illustrate the characterization of in vitro T cell subset expansion and proliferation in PBMCs collected from EBV-infected donor D in response to the CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated substituted anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibody was tested at indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A–9B), EBV-specific Tcm CD8+ cells (Figures 9C–9D), and EBV-specific Tem CD8+ cells (Figures 9E–9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 13-1] Figures 13A–13D show the time course (days) of tumor volume (Figure 13A) and body weight (Figure 13B) in NSG mice with ZR-75-1 tumors implanted with human CD3+ T cells expanded in vitro. Groups of 10 mice were treated with either a vehicle or a Her2 / CD28×CD3 trispecific antibody at the indicated doses. The arrowheads indicate the day of administration. Tumor volume is depicted as mean ± SEM, mm3. Body weight change is depicted as change%, mean ± SEM. The X-axis shows the number of days after ZR-75-1 cell implantation. Figure 13C shows the time course of tumor volume (mm3) per individual mouse in each treatment group. Figure 13D shows the tumor weight (mg) for each treatment group. **=p<0.001; ***=p<0.0003 (two-way ANOVA, control vs. 100 and 10 ug / kg). [Figure 13-2] Continuation of Figure 13-1. [Figure 13-3] Continuation of Figure 13-2. [Figure 13-4] Continuation of Figure 13-3. [Figure 14-1] Figures 14A–14C show the effects of Her2 / CD28×CD3 trispecific antibody treatment on T cells from whole blood. Figure 14A shows flow cytometry analysis of hCD45+, CD8+, CD4+, and mCD45+ cells. Figure 14B shows the effects of control or Her2 / CD28×CD3 trispecific antibody treatment (at indicated doses) on hCD45+, CD8+, CD4+, and mCD45+ cell counts. Figure 14C shows the effects of control or Her2 / CD28×CD3 trispecific antibody treatment (at indicated doses) on human cell ratios (CD4+ / CD45+ and CD8+ / CD45+). For each x-axis parameter shown in Figures 14B and 14C, the conditions are (from left to right): control, 100 ug / kg of trispecific antibody, 10 ug / kg of trispecific antibody, 1 ug / kg of trispecific antibody, and 0.1 ug / kg of trispecific antibody. The percentages shown in Figures 14B and 14C are based on the control sample versus 100 ug / kg. [Figure 14-2] Continuation of Figure 14-1. [Figure 15-1] Figures 15A–15C show the effects of Her2 / CD28×CD3 trispecific antibody treatment on tumor-infiltrating lymphocytes (TILs) as examined by immunohistochemistry (IHC). Arrows indicate tumor-infiltrating T cells identified in ZR-75-1 mammary tumors. The upper images are at 1× magnification; the lower images are at 20× magnification. In both sets of images, staining for human CD45, human CD4, and human CD8 is shown from left to right. Tumors from mice treated with vehicle control (Figure 15A), 100 ug / kg of trispecific antibody (Figure 15B), or 0.1 ug / kg of trispecific antibody (Figure 15C) are shown. [Figure 15-2] Continuation of Figure 15-1. [Figure 15-3] Continuation of Figure 15-2. [Figure 16-1]Figures 16A–16C show the quantitative effects of Her2 / CD28×CD3 trispecific antibody treatment on TILs as measured by IHC. Each dot represents one tumor from an individual mouse; rectangles represent the group mean; error bars indicate the standard deviation. * = p < 0.05 (ANOVA) compared to the vehicle control group. The number of CD45+ (Figure 16A), CD4+ (Figure 16B), or CD8+ (Figure 16C) cells is shown. In Figure 16C, due to excessive nonspecific signaling in the CD8 IHC slide, a region quantification approach was used for CD8+ cells instead of a cell counting algorithm. [Figure 16-2] Continuation of Figure 16-1. [Figure 16-3] Continuation of Figure 16-2. [Figure 17-1] Figures 17A–17F show in vitro cytolysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by a Her2 / CD28×CD3 trispecific antibody ("ctl") that has a wild-type trastuzumab antigen-binding domain and an anti-CD3 antigen-binding domain and does not have the 32 / 35QQ mutation in the VL domain, compared with a Her2 / CD28×CD3 trispecific antibody having mutations in the VL domains of the anti-HER2 and anti-CD3 arms (numbered as shown in Table 1). Cytotoxic activity against cell lines with various HER2 expression levels is depicted: HCC1954 (Figure 17A) for high HER2 expression, BT20 (Figure 17C) for moderate HER2 expression, and MDA-MD-231 (Figure 17E) for low HER2 expression. Graphs are shown illustrating cell elimination as a function of antibody concentration against target cells HCC1954 (Figure 17B), BT20 (Figure 17D), and MDA-MD-231 (Figure 17F), comparing binding protein #2 with a control or binding proteins #1 and #5 with a control. [Figure 17-2] Continuation of Figure 17-1. [Figure 17-3] Continuation of Figure 17-2. [Figure 17-4] Continuation of Figure 17-3. [Figure 17-5] Continuation of Figure 17-4. [Figure 17-6]Continuation of Figure 17-5. [Figure 18-1] Figures 18A and 18B summarize the mean EC50 (pM) of in vitro cell killing with experimental or control Her2 / CD28×CD3 trispecific antibodies against the indicated breast cancer (Figure 18A) or gastric cancer cell lines (Figure 18B). The amino acid sequences of the indicated trispecific antibodies are provided in Table 1. [Figure 18-2] Continuation of Figure 18-1. [Modes for carrying out the invention]

[0046] This disclosure provides a triplicate and / or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein the first pair of polypeptides forming the binding protein has a crossover-oriented bivariate domain.

[0047] general definition When used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated. Unless otherwise required by context, singular terms include plural terms, and plural terms include singular terms.

[0048] It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "include," "consist of," and "essentially consist of."

[0049] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer with a length of at least 10 nucleotides. In certain embodiments, the nucleotides constituting the polynucleotide may be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications, e.g., bromouridine; ribose modifications, e.g., arabinosides and 2',3'-dideoxyribose; and internucleotide ligation modifications, e.g., phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladetes, and phosphoramidates. The term "polynucleotide" particularly includes single-stranded and double-stranded forms of DNA.

[0050] "Isolated polynucleotide" means a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, which (1) is not associated with any or all polynucleotides found in nature, (2) is linked to a polynucleotide that is not linked in nature, or (3) does not exist in nature as a part of a larger sequence.

[0051] An isolated polypeptide is a polypeptide that (1) is free from at least some other polypeptides normally found with it, (2) is essentially free from other polypeptides from the same source, e.g., from the same species, (3) is expressed by cells from different species, (4) is isolated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other material that associate with it in nature, (5) is not associated (by covalent or non-covalent interactions) with any portion of the polypeptide that associates with it in nature, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide that does not associate with it in nature, or (7) is not present in nature. Such an isolated polypeptide can be encoded by synthetically derived genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof. Preferably, the isolated polypeptide is substantially free from polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0052] Naturally occurring antibodies typically contain tetramers. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50–70 kDa). The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity to a target antigen. The amino-terminal portions of each light and heavy chain typically contain a variable domain of about 100–110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portions of each chain typically define a constant domain, which is responsible for effector function. Thus, in naturally occurring antibodies, full-length heavy-chain immunoglobulin polypeptides have a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3) including, V H The domain is located at the amino terminus of the polypeptide, C H3 The domain is located at the carboxyl terminus, and the full-length light chain immunoglobulin The lipeptide has a variable domain (V L ) and constant domain (C L ) including, V L The domain is located at the amino terminus of the polypeptide, C L The domain is located at the carboxyl terminus.

[0053] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly further divided into subclasses, including but not limited to IgA1 and IgA2. Within the full-length light and heavy chains, variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 further amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989) (incorporated as a whole for all purposes by reference). The variable regions of each light / heavy chain pair typically form antigen-binding sites. The variable domains of naturally occurring antibodies typically exhibit the same overall structure: a relatively conserved framework region (FR) joined by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework region, which can enable binding to specific epitopes. From the amino-terminus to the carboxyl-terminus, the variable domains of both the light and heavy chains typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0054] The term "CDR set" refers to a group of three CDRs present within a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently according to different systems. (Kabat et al., SEQUENCES) The system described by *Of Proteins of Immunological Interest* (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs can be referred to as Kabat CDRs. (Chothia and Lesk, 1987, J. Mol. Biol. 196: pp. 901-917; Chothia et al.) Al. (1989, Nature 342:877-83) found that certain sub-regions within the Kabat CDR adopt nearly identical peptide skeletal conformations despite exhibiting significant diversity at the amino acid sequence level. These sub-regions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR are described by Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Furthermore, other CDR boundary definitions may not strictly adhere to one of the systems herein, but nevertheless overlap with Kabat CDRs, however they may be shortened or lengthened in light of predictions or experimental findings that specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding. Methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia-defined CDRs. The identification of predicted CDRs using amino acid sequences is described by Martin, A. This is well known in the field, as seen in C., "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2, edited by Kontermann R. and Dubel S., Springer-Verlag, Berlin, pp. 33-51 (2010). Other conventional methods allow for the identification of CDR sequences by examining the amino acid sequences of heavy and / or light chain variable domains, for example, by determining hypervariable regions by comparing them with known amino acid sequences of other heavy and light chain variable regions. Numbered sequences can be aligned visually or using alignment programs, such as one of the CLUSTAL suite programs described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models have traditionally been used to accurately depict the framework and CDR region, and therefore, accurate sequence-based assignments.

[0055] When used herein, the term "Fc" refers to a molecule comprising a sequence of non-antigen-binding fragments, whether monomeric or polymeric, resulting from or produced by the digestion of an antibody, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins, but IgG1 and IgG2 are preferred. Fc molecules consist of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent associations to form dimeric or polymeric forms. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of Fc is a disulfide-bonded dimer resulting from the papain digestion of IgG. When used herein, the term “native Fc” is comprehensive to monomeric, dimeric, and polymeric forms.

[0056] The F(ab) fragment typically consists of one light chain and one heavy chain. H and C H1 Includes the domain and V of the F(ab) fragment H -C H1 The heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. As used herein, the F(ab) fragment also contains one light chain containing two variable domains separated by an amino acid linker, and two variable domains separated by an amino acid linker and C H1 It may contain a single heavy chain that includes a domain.

[0057] The F(ab') fragment typically consists of one light chain and (C H1 and C H2 The molecule contains a portion of the heavy chain that includes a further constant region (between the domains), and as a result, an interchain disulfide bond can be formed between the two heavy chains to form an F(ab')2 molecule.

[0058] The term “binding protein,” as used herein, refers to a molecule that does not exist naturally (or is recombinant or engineered) and specifically binds to at least one target antigen. Unless otherwise specified, the triplicate binding proteins of this disclosure typically comprise four polypeptide chains forming at least three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It has a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It has a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 [III] It has a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It has a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C LThis is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; The polypeptides of formula I and formula II form a crossover light-heavy chain pair.

[0059] A "recombinant" molecule is a molecule that has been manufactured, expressed, created, or isolated by recombinant means.

[0060] One embodiment of the present disclosure provides a binding protein having biological and immunological specificity for one to three target antigens. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

[0061] The term "swappability," as used herein, refers to the interchangeability of variable domains within a binding protein format, and with respect to the preservation of folding and ultimate binding affinity. "Full swapability" refers to the interchangeability of variable domains in a polypeptide chain of formula I or a polypeptide chain of formula II while maintaining the full functionality of the binding protein as demonstrated by the preservation of binding affinity. H1 and V H2 The order of both domains, and therefore V L1 and V L2 This refers to the ability to swap the order of domains (i.e., reverse the order). Furthermore, VH and V L It should be noted that the designation merely refers to the location of a domain on a particular protein chain in the final format. For example, V H1 and V H2 V in the parent antibody L1 and V L2 It can originate from the domain, and V in binding proteins H1 and V H2 It can be placed in position. Similarly, V L1 and V L2 V in the parent antibody H1 and V H2 It can originate from the domain, and V in binding proteins H1 and V H2 It can be placed in position V H and V L This designation refers to the current position, not the original position within the parent antibody. H and V L Domains are therefore "swappable".

[0062] The terms “antigen,” “target antigen,” or “antigen target,” as used herein, refer to a molecule or portion of a molecule to which a binding protein can bind and which can be used in animals to produce an antibody that can bind to the epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a protein, the binding protein can compete with the intact antibody that recognizes the target antigen.

[0063] The term "Her2" refers to the human epidermal growth factor receptor 2, a member of the epidermal growth factor receptor family.

[0064] "CD3" is a differentiation antigen group factor 3 polypeptide and is typically a T cell surface protein that is part of the T cell receptor (TCR) complex.

[0065] "CD28" is a differentiation antigen group 28 polypeptide and a T cell surface protein that provides costimulatory signals for T cell activation and survival.

[0066] "CD38" is a differentiation antigen group 38 polypeptide and is a glycoprotein found on the surface of many immune cells.

[0067] The term "T cell engager" refers to binding proteins that are directed not only to the host immune system, and more specifically to the cytotoxic activity of T cells, but also to tumor target proteins.

[0068] The term "single-specific binding protein" refers to a binding protein that specifically binds to a single antigen target.

[0069] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[0070] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

[0071] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0072] The term "triple-specific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

[0073] The term "trivalent binding protein" refers to a binding protein having three binding sites. In certain embodiments, a trivalent binding protein can bind to one antigen target. In other embodiments, a trivalent binding protein can bind to two antigen targets. In other embodiments, a trivalent binding protein can bind to three antigen targets.

[0074] "Isolated" binding proteins are binding proteins identified and separated and / or recovered from components of their natural environment. Contaminating components of their natural environment are materials that interfere with the diagnostic or therapeutic use of binding proteins and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, binding proteins are purified to (1) a higher than 95% by weight, most preferably higher than 99% by weight, by Lowry determination; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to a homogeneous state by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Since at least one component of the binding protein's natural environment is absent, the isolated binding protein and One example is in situ binding proteins within recombinant cells.

[0075] The terms “substantially pure” or “substantially purified,” as used herein, refer to a compound or species that is the dominant species present (i.e., it is present in greater abundance than any other individual species in the composition, based on molar concentration). In some embodiments, a substantially purified fraction is a composition in which a species constitutes at least about 50% (based on molar concentration) of all polymer species present. In other embodiments, a substantially pure composition is a composition in which all polymer species present in the composition constitute more than about 80%, 85%, 90%, 95%, or 99%. In yet another embodiment, the species are purified to an intrinsically homogeneous state in which the composition is essentially composed of a single polymer species (impurity species cannot be detected in the composition by conventional detection methods).

[0076] The term “epitope” includes any determinant, preferably polypeptide determinant, that can specifically bind to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinant includes a chemically active surface grouping of a molecule, such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments, may have distinctive three-dimensional structural features and / or distinctive electrical features. An epitope is a region of an antigen to which an antibody or binding protein binds. In certain embodiments, a binding protein is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the equilibrium dissociation constant is ≤10 -8 When M is the case, more preferably the equilibrium dissociation constant is ≤10 -9 When M is the case, the most preferable dissociation constant is ≤10 -10 When the protein is M, it is said that the binding protein specifically binds to the antigen.

[0077] Dissociation constant (K) of binding proteins D This can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligands (target antigens on a biosensor matrix) and analytes (binding proteins in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing analytes (binding proteins on a biosensor matrix) and presenting ligands (target antigens). D When used herein, the term "dissociation constant" refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0078] When used herein, the term "specifically binds" means approximately 1 × 10⁻⁶ -6 M, 1×10 -7 M, 1×10 -8 M, 1×10-9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 This refers to the ability of a binding protein or antigen-binding fragment to bind to an antigen containing an epitope with M or less Kd, and / or to bind to the epitope with an affinity at least twice as high as its affinity to a nonspecific antigen.

[0079] In some embodiments, the antigen-binding domain and / or binding protein of this disclosure "cross-reacts" with human and cynomolgus monkey CD38 polypeptides, e.g., CD38 extracellular domain, human CD38 isoform A, human CD38 isoform E, and cynomolgus monkey CD38. 50 The binding of the binding protein to antigen 1 (Ag1) is "cross-reactive" to antigen 2 (Ag2) when the affinity values ​​for both antigens are within similar ranges. In this application, the binding of the binding protein to Ag1 is cross-reactive to Ag2 when the affinity ratio of Ag2 to Ag1 is equal or less than 20, where the affinity is measured using the same method for both antigens.

[0080] The term "linker" as used herein refers to immunoglobulin May A linker refers to one or more amino acid residues inserted between the domains, providing sufficient mobility to the light and heavy chain domains for folding into a crossover bivariate domain immunoglobulin. Linkers are inserted at the sequence level, at transitions between variable domains or between variable and constant domains. Since the approximate size of immunoglobulin domains is well understood, transitions between domains can be identified. The precise location of domain transitions can be determined by positioning secondary structure elements, such as beta sheets or peptide stretches that do not form alpha-helices, as demonstrated by experimental data or estimated by modeling or secondary structure prediction techniques. The linkers described herein are V L2C-terminus and V of the domain L1 L1 and V are located on the light chain between the N-terminuses of the domain. L1 C-terminus and C L It is called L2, located on the light chain between the N-terminuses of the domain. The heavy chain linker is V H1 C-terminus and V of the domain H2 L3 and V are located between the N-terminuses of the domain. H2 C-terminus and C H1 It is known as L4, located between the N-terminuses of a domain.

[0081] The term “vector,” as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transmit coding information to a host cell. The term “vector” includes nucleic acid molecules that can transport another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which an additional DNA segment can be inserted. Another type of vector is a viral vector, in which an additional DNA segment can be inserted into the viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Additionally, certain vectors can dictate the expression of a gene to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vector, the terms “plasmid” and “vector” may be used interchangeably herein. However, this disclosure is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0082] The term “recombinant host cell” (or “host cell”), as used herein, refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to a specific target cell but also to the offspring of such cells. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in successive generations due to either mutation or environmental influences; however, such cells are still included within the scope of the term “host cell” as used herein. A variety of host cell expression systems can be used to express binding proteins, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). A suitable example of a bacterial expression vector is pUC19. To express a binding protein by recombination, host cells are transformed or transfected with one or more recombinant expression vectors having DNA fragments encoding the polypeptide chain of the binding protein, resulting in the expression of the polypeptide chain in the host cell, preferably secreted into the culture medium in which the host cell is cultured, from which the binding protein can be recovered.

[0083] When used herein, the term "transformation" refers to the transformation of the genetic characteristics of a cell. Transformation refers to a change in a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed, genetically modified from its native state. After transformation, the transforming DNA can undergo recombination with the cell's DNA by being physically incorporated into the cell's chromosomes, or it can be transiently maintained as an episomal element without replication, or it can replicate independently as a plasmid. A cell is considered stably transformed if the DNA replicates with cell division. The term "transfection," as used herein, refers to the uptake of exogenous or extrinsic DNA by a cell, and a cell is "transfected" when extrinsic DNA is introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Using such techniques, one or more extrinsic DNA molecules can be introduced into a suitable host cell.

[0084] The term "naturally occurring," as used herein and applied to an object, refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, polynucleotides or polypeptides present in organisms (including viruses) that can be isolated from a natural source and have not been intentionally modified by humans are naturally occurring. Similarly, "not naturally occurring," as used herein, refers to an object that is not found in nature or has been structurally modified or synthesized by humans.

[0085] Where used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs, e.g., α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptide chains of binding proteins. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In polypeptide notation used herein, according to standard usage and convention, the left-handed direction is towards the amino terminus and the right-handed direction is towards the carboxyl terminus.

[0086] Naturally occurring residues can be classified into classes based on their common side-chain characteristics: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basicity: His, Lys, Arg; (8) Residues that affect chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0087] Conservative amino acid substitutions may involve the exchange of one member of one class with another member of the same class. Non-conservative substitutions may involve the exchange of one member of one class with a member of another class.

[0088] Those skilled in the art can determine preferred variants of a polypeptide chain of a binding protein using well-known techniques. For example, they may identify preferred segments of a polypeptide chain that can be altered without disrupting activity by targeting regions that are not considered important for activity. Alternatively, they may identify conserved residues and molecular segments among similar polypeptides. In addition, segments that may be important for biological activity or structure can be subjected to conserved amino acid substitutions without disrupting biological activity or adversely affecting the polypeptide structure.

[0089] The term "patient" as used herein includes both human and animal subjects.

[0090] The terms “treatment” or “to treat” as used herein refer to both therapeutic treatments and prophylactic or preventative measures. Persons requiring treatment include those with a disability, those predisposed to a disability, or those for whom a disability should be prevented. In certain embodiments, binding proteins can be used to treat a person with cancer or a person susceptible to cancer, or to induce remission of cancer in a human subject. Binding proteins can also be used to prevent cancer in a human patient. In certain embodiments, cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer, e.g., Her2+ breast cancer, germinal center B-cell lymphoma, or B-cell acute lymphoblastic leukemia, and in other embodiments, binding proteins can be used to treat a person with an inflammatory disorder or a person susceptible to an inflammatory disorder, or to induce remission of an inflammatory disorder in a human subject.

[0091] When used herein, the terms "pharmaceutical composition" or "therapeutic composition" refer to a compound or composition that, when appropriately administered to a patient, can induce a desired therapeutic effect.

[0092] The terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier,” as used herein, refer to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

[0093] The terms “effective dose” and “therapeutic dose,” when used in reference to a pharmaceutical composition containing one or more binding proteins, refer to a quantity or dosage sufficient to produce the desired therapeutic outcome. More specifically, the therapeutic dose is the amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over a period of time. The effective dose may vary depending on the specific binding protein being used, as well as on various factors and conditions relating to the patient being treated, and the severity of the disorder. For example, when the binding protein is administered in vivo, factors to be considered include the patient's age, weight, and health, as well as dose-response curves and toxicity data obtained in preclinical animal studies. Determining the effective dose or therapeutic dose of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0094] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of binding protein.

[0095] Triple-specific and / or trivalent binding proteins Certain aspects of the present disclosure include a triplicate and / or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, the first of which polypeptides form the binding protein The present invention relates to a binding protein in which the pair has a bivariate domain having crossover orientation, and the second pair of polypeptides forming the binding protein has a single variable domain. Any CDR or variable domain of any antigen-binding protein described herein may have an application in the triplicate binding protein of this disclosure.

[0096] In some embodiments, each of the three antigen-binding sites binds to a different target (e.g., a polypeptide antigen). In some embodiments, the triplicate binding protein comprises four polypeptide chains that form three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 and C H2 It is an immunoglobulin hinge region that connects domains; L1, L2, L3, and L4 are amino acid linkers; The polypeptides of formula I and formula II form a crossover light-heavy chain pair.

[0097] Any antigen-binding site described herein is intended to have use in, for example, the tripspecific binding protein of this disclosure, which comprises four polypeptide chains having the above structure. For example, in some embodiments, the tripspecific binding protein of this disclosure has a first antigen-binding site V H1 and V L1 The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 Domain pair and V that form a third antigen-binding site H3 and V L3 Includes a domain pair. In some embodiments, the triplicate binding protein of this disclosure forms a first antigen-binding site that binds to the CD28 polypeptide. H1 and V L1 The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 Domain pair and V that form a third antigen-binding site H3 and V L3 Includes a domain pair. In some embodiments, the triplicate binding protein of this disclosure forms a first antigen-binding site V H1 and V L1The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 The domain pair and the V form a third antigen-binding site that binds to tumor target proteins. H3 and V L3 Includes domain pairs. Some embodiments In this invention, the triple-specific binding protein forms a first antigen-binding site that binds to the CD28 polypeptide. H1 and V L1 The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 The domain pair and the V form a third antigen-binding site that binds to tumor target proteins. H3 and V L3 Includes a domain pair. In some embodiments, the triplicate binding protein of this disclosure forms a first antigen-binding site that binds to the CD28 polypeptide. H1 and V L1 The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 The domain pair and the V form a third antigen-binding site that binds to the CD38 polypeptide. H3 and V L3 Includes a domain pair. In some embodiments, the triplicate binding protein of this disclosure forms a first antigen-binding site that binds to the CD28 polypeptide. H1 and V L1 The domain pair forms a second antigen-binding site that binds to the CD3 polypeptide. H2 and V L2 V forms a domain pair and a third antigen-binding site that binds to the HER2 polypeptide. H3 and V L3 Includes domain pairs.

[0098] In some embodiments, the binding proteins of this disclosure bind to one or more tumor target proteins and one or more T cell target proteins. In some embodiments, the binding proteins can specifically bind to one tumor target protein and two different epitopes on a single T cell target protein. In some embodiments, the binding proteins can specifically bind to one tumor target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen-binding site that specifically binds to the tumor target protein. In some embodiments, the target protein is CD38 or HER2. Additional tumor target proteins are provided below. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28. Exemplary and non-limiting polypeptides that may have applications in any of the triplicate binding proteins described herein are provided in Table 1.

[0099] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 156 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 157 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 158 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 159 by at least 95%.

[0100] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 160 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 161 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 162 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 163 by at least 95%.

[0101] In some embodiments, the binding protein of this disclosure forms three antigen-binding sites. The molecule comprises four polypeptide chains, the first polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 167.

[0102] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 168 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 169 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 170 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 171 by at least 95%.

[0103] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 172 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 173 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 174 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 175 by at least 95%.

[0104] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 176 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 177 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 178 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 179 by at least 95%.

[0105] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 181 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 182 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 183 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 184 by at least 95%.

[0106] In some embodiments, the binding protein of this disclosure forms three antigen-binding sites. It comprises four polypeptide chains, the first polypeptide chain having an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 185 or the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain having an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 186 or the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain having an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 187 or the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain having an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 188 or the amino acid sequence of SEQ ID NO: 188.

[0107] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 100 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 101 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 102 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 103 by at least 95%.

[0108] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 104 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 105 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 106 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 107 by at least 95%.

[0109] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 112 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 113 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 114 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 115 by at least 95%.

[0110] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 116 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 117 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 118 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 119 by at least 95%.

[0111] In some embodiments, the binding protein of this disclosure forms three antigen-binding sites. The molecule comprises four polypeptide chains, the first polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 123.

[0112] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 124 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 125 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 126 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 127 by at least 95%.

[0113] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 131.

[0114] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 132 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 133 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 134 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 135 by at least 95%.

[0115] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises an amino acid sequence identical by at least 95% to the amino acid sequence of SEQ ID NO: 136 or the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises an amino acid sequence identical by at least 95% to the amino acid sequence of SEQ ID NO: 137 or the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises an amino acid sequence identical by at least 95% to the amino acid sequence of SEQ ID NO: 138 or the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises an amino acid sequence identical by at least 95% to the amino acid sequence of SEQ ID NO: 139 or the amino acid sequence of SEQ ID NO: 139.

[0116] In some embodiments, the binding protein of this disclosure forms three antigen-binding sites. The molecule comprises four polypeptide chains, the first polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 143.

[0117] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 144 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 145 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 146 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 147 by at least 95%.

[0118] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 148 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 149 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 150 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 151 by at least 95%.

[0119] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 152 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 153 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 154 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 155 by at least 95%.

[0120] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 286 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 287 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 288 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 288 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 289 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 289 by at least 95%.

[0121] In some embodiments, the binding protein of this disclosure forms three antigen-binding sites. The molecule comprises four polypeptide chains, the first polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 290; the second polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 291; the third polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 292; and the fourth polypeptide chain containing at least 95% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 293.

[0122] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 294 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 294 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 295 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 295 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 296 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 296 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 297 by at least 95%.

[0123] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 298 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 298 by at least 95%; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 299 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 299 by at least 95%; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 300 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 300 by at least 95%; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 301 or an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 301 by at least 95%.

[0124] Anti-CD38 binding site Certain aspects of this disclosure relate to binding proteins containing an antigen-binding site that binds to a CD38 polypeptide. In some embodiments, the CD38 polypeptide is a human CD38 polypeptide also known as ADPRC1. Human CD38 polypeptides are known in the Art, but are not limited to, the polypeptide represented by NCBI accession number NP_001766.2, or the polypeptide produced from NCBI gene identification number 952. In some embodiments, the antigen-binding site binds to a human CD38 polypeptide, a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide, or a combination of human CD38 polypeptide and a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide. In some embodiments, the binding protein containing an antigen-binding site that binds to a CD38 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, triplicate and / or trivalent, or multispecific and / or polyvalent.

[0125] In some embodiments, either the CDR and / or variable domain of the anti-CD38 binding site described later may be used in a monospecific antibody.

[0126] In other embodiments, either the CDR and / or variable domain of the anti-CD38 binding site described below may be used at any of the binding sites of a triple-specific binding protein comprising, for example, four polypeptides that form the three antigen-binding sites described above. In certain embodiments, the antigen-binding site includes a CD38 polypeptide. The binding protein is a triple-specific binding protein containing four polypeptides that form the three antigen-binding sites described above, V H3 and V L3 The pair of domains forms a third antigen-binding site that binds to the CD38 polypeptide.

[0127] Various exemplary binding sites and binding proteins are described herein. For example, in some embodiments, the anti-CD38 binding site cross-reacts with human CD38 (e.g., human CD38 isoform A and / or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, the binding protein containing the anti-CD38 binding site induces apoptosis in CD38+ cells. In some embodiments, the binding protein containing the anti-CD38 binding site recruits T cells to CD38+ cells and optionally activates the T cells (e.g., via TCR stimulation and / or co-stimulation).

[0128] In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).

[0129] In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence containing the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence containing the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24).

[0130] In some embodiments, the binding site to CD38 is an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR-H2 sequence containing the amino acid sequence of INPGSGST (SEQ ID NO: 26), and a CDR-H3 sequence containing the amino acid sequence of ARYAYGY (SEQ ID NO: 27); and / or a CDR-L1 sequence containing the amino acid sequence of QNVGTA (SEQ ID NO: 28), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 29), and QQYSTYPFT (sequence number The antibody light chain variable (VL) domain includes a CDR-L3 sequence containing the amino acid sequence of (SEQ ID NO. 30). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing a CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO. 25), a CDR-H2 sequence containing the amino acid sequence of INPGSGST (SEQ ID NO. 26), and a CDR-H3 sequence containing the amino acid sequence of ARYAYGY (SEQ ID NO. 27); and an antibody light chain variable (VL) domain containing a CDR-L1 sequence containing the amino acid sequence of QNVGTA (SEQ ID NO. 28), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO. 29), and a CDR-L3 sequence containing the amino acid sequence of QQYSTYPFT (SEQ ID NO. 30).

[0131] In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence containing the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36). In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence containing the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36).

[0132] In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence containing the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence containing the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42).

[0133] In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence containing the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). In some embodiments, the binding site to CD38 includes GFTFSS The antibody heavy chain variable (VH) domain includes a CDR-H1 sequence containing the amino acid sequence of YG (SEQ ID NO: 43), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence containing the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and an antibody light chain variable (VL) domain including a CDR-L1 sequence containing the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48).

[0134] In some embodiments, the binding site that binds to CD38 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 79 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 80. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 79 and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 80.

[0135] In some embodiments, the binding site that binds to CD38 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 81 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 82. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 81, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 82.

[0136] In some embodiments, the binding site that binds to CD38 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 83 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 84. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 83, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 84.

[0137] In some embodiments, the binding site that binds to CD38 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 87, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 88. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 87, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 88.

[0138] In some embodiments, the binding site that binds to CD38 is, A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 89 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 90. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 89, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 90.

[0139] In some embodiments, the binding site that binds to CD38 is, A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 85, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 86. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 85, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 86.

[0140] In some embodiments, the binding site that binds to CD38 is, QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTLTVSS(Sequence No. 2) 77) A variable antibody heavy chain (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the above, and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO: 278). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 277 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 278. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 277 and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 278. In some embodiments, the VH and / or VL domains are humanized.

[0141] In some embodiments, the binding site that binds to CD38 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSMDYWGQGTSVTVSS (SEQ ID NO: 279), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK (SEQ ID NO: 280). In some embodiments, the binding site to CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 279 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 280. In some embodiments, the binding site for CD38 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 279 and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 280. In some embodiments, the VH and / or VL domains are humanized.

[0142] In some embodiments of the above embodiments, the binding protein is a triple-specific binding protein. In some embodiments, the triple-specific binding protein includes an antigen-binding site that binds to a CD38 polypeptide, an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a triple-specific binding protein comprising four polypeptides, each containing three antigen-binding sites, and the polypeptide of formula I and the polypeptide of formula II are , forming a crossover light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above-described anti-CD38 antigen-binding sites are V H3 and V L3 This represents a third antigen-binding site that binds to the CD38 polypeptide. In some embodiments, VH1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, and the VH and VL domains of either of the anti-CD38 antigen-binding sites described above and / or in Table 2 are V H3 and V L3 This represents a third antigen-binding site that binds to the CD38 polypeptide.

[0143] Exemplary sequences of anti-CD38 antigen-binding sites are provided in Table 2. In some embodiments, the binding protein containing the anti-CD38 antigen-binding site of this disclosure contains all of the CDR sequences 1, 2, 3, 4, 5, or 6 of the anti-CD38 antibody listed in Table 2. In some embodiments, the binding protein containing the anti-CD38 antigen-binding site of this disclosure contains the VH domain sequence and / or VL domain sequence of the anti-CD38 antibody listed in Table 2.

[0144] [Table 1] [Table 2] [Table 3]

[0145] Furthermore, antibodies (e.g., monospecific antibodies) comprising either the anti-CD38 CDR and / or variable domain described above are provided herein.

[0146] In some embodiments, the binding proteins of this disclosure include antigen-binding sites that bind to the extracellular domain of human CD38 polypeptide and the extracellular domain of cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether the antigen-binding site binds to an antigen are described herein and are known in the art, including, but are not limited to, ELISA, SPR, and flow cytometry assays.

[0147] Anti-HER2 binding site Certain aspects of this disclosure relate to binding proteins containing antigen-binding sites that bind to HER2 polypeptides. In some embodiments, the HER2 polypeptide is a human HER2 polypeptide also known as NEU, NGL, ERBB2, TKR1, CD340, HER-2, MLN19, and HER-2 / neu. Human HER2 polypeptides are known in the art, but are not limited to, polypeptides represented by NCBI accession numbers XP_024306411.1, XP_024306410.1, XP_024306409.1, NP_001276867.1, NP_001276866.1, NP_001276865.1, NP_001005862.1, or NP_004439.2, or polypeptides produced from NCBI gene identification number 2064. In some embodiments, the binding protein containing the antigen-binding site that binds to the HER2 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or polyvalent. In some embodiments, the binding protein containing the antigen-binding site that binds to the HER2 polypeptide is a trispecific binding protein containing four polypeptides that form the three antigen-binding sites described above, and V H3 and V L3 The domain pair forms a third antigen-binding site that binds to the HER2 polypeptide.

[0148] In some embodiments, the binding site for HER2 is a CDR-H1 sequence containing the amino acid sequence GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPT The antibody heavy chain variable (VH) domain includes a CDR-H2 sequence containing the amino acid sequence of NAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain including a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0149] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0150] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0151] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0152] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0153] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0154] In some embodiments, the binding site for HER2 is the CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), and the IYPTNGYT (SEQ ID NO: 3) The antibody heavy chain variable (VH) domain includes a CDR-H2 sequence containing a amino acid sequence and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and / or an antibody light chain variable (VL) domain including a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0155] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0156] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0157] In some embodiments, the binding site for HER2 is the CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), and the IYPTQGYT (SEQ ID NO: 4) The antibody heavy chain variable (VH) domain includes a CDR-H2 sequence containing a amino acid sequence and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain including a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0158] In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0159] In some embodiments, the binding site that binds to HER2 is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (Sequence ID 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (distribution For the amino acid sequences of (sequence number 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (sequence number 75), or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (sequence number 76), at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, and less Antibody heavy chain variable (VH) domains containing at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76; and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78.

[0160] In some embodiments, the binding site that binds to HER2 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site for HER2 contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77.

[0161] In some embodiments, the binding site that binds to HER2 is For the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and at least 91%. Antibody heavy chain variable (VH) domains containing %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences, and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site for HER2 contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 73, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 73, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77.

[0162] In some embodiments, the binding site that binds to HER2 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site for HER2 contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 75, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 75, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77.

[0163] In some embodiments, the binding site that binds to HER2 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), and / or DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 74, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 74, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77.

[0164] In some embodiments, the binding site that binds to HER2 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site for HER2 contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 76, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 76, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77.

[0165] In some embodiments, the binding site that binds to HER2 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or For the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78), at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, and less than 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%. The antibody light chain variable (VL) domain contains at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 78. In some embodiments, the binding site for HER2 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 78.

[0166] In some embodiments, the anti-HER2 antigen-binding site of the Disclosure includes one, two, three, four, five, or all six CDR sequences of the anti-HER2 antibody trastuzumab, 30R / 55Q / 102E, 30R / 56A / 102S, 30R / 55Q / 102S, 30R / 56A / 102E, or 30Q. In some embodiments, the anti-HER2 antigen-binding site of the Disclosure includes the VH domain sequence and / or VL domain sequence of the anti-HER2 antibody trastuzumab, 30R / 55Q / 102E, 30R / 56A / 102S, 30R / 55Q / 102S, 30R / 56A / 102E, or 30Q.

[0167] Exemplary anti-HER2 antigen-binding site sequences are provided in Table 3. In some embodiments, the anti-HER2 antigen-binding site of this disclosure comprises all of the CDR sequences 1, 2, 3, 4, 5, or 6 of the anti-HER2 antibody listed in Table 3. In some embodiments, the anti-HER2 antigen-binding site of this disclosure comprises the VH domain sequence and / or VL domain sequence of the anti-HER2 antibody listed in Table 3.

[0168] [Table 4] [Table 5]

[0169] Other antitumor target binding sites In some embodiments, the binding protein of this disclosure includes an antigen-binding site that binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (e.g., human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., human HER2 polypeptide).In some embodiments, the tumor target proteins of this disclosure include A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), and CCL8 (mcp CCL11 (also known as MIP-2), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (FCER2, as an IgE receptor) CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (as ICOS) (also known as PD-1), CD279, CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP. FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, L Examples of antigen targets include, but are not limited to, PFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0170] Anti-CD28 binding site Certain aspects of this disclosure relate to binding proteins containing an antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide also known as Tp44. Human CD28 polypeptides are known in the Art, but are not limited to, polypeptides represented by NCBI accession numbers XP_011510499.1, XP_011510497.1, XP_011510496.1, NP_001230007.1, NP_001230006.1, or NP_006130.1, or polypeptides produced from NCBI gene identification number 940. In some embodiments, the binding protein containing an antigen-binding site that binds to a CD28 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, triplicate and / or trivalent, or multispecific and / or polyvalent. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD28 polypeptide is a triple-specific binding protein containing four polypeptides that form three antigen-binding sites. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD28 polypeptide is a triple-specific binding protein containing four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein containing four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide, and one of which binds to the CD38 polypeptide. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein containing four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide, and one of which binds to the HER2 polypeptide.In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein containing four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide, and one of which binds to the tumor target protein.

[0171] In some embodiments, the binding site that binds to CD28 is GYTFTSYY(sequence). The antibody heavy chain variable (VH) domain includes a CDR-H1 sequence containing the amino acid sequence of (number 49), a CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (sequence number 50), and a CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (sequence number 51), and / or an antibody light chain variable (VL) domain including a CDR-L1 sequence containing the amino acid sequence of QNIYVW (sequence number 52), a CDR-L2 sequence containing the amino acid sequence of KAS (sequence number 53), and a CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (sequence number 54). In some embodiments, the binding site for CD28 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and a CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53), and a CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (SEQ ID NO: 54).

[0172] In some embodiments, the binding site that binds to CD28 is, An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92). In some embodiments, the binding site to CD28 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 91, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 92. In some embodiments, the binding site for CD28 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 91, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 92.

[0173] In some embodiments of the above embodiments, the binding protein is a triple-specific binding protein. In some embodiments, the triple-specific binding protein includes an antigen-binding site that binds to a tumor target protein (but not limited to, CD38 or HER2), an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a triple-specific binding protein comprising four polypeptides, each containing three antigen-binding sites, where the polypeptide of formula I and the polypeptide of formula II form a crossover light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above-described anti-CD28 antigen-binding sites are V H1 and V L1 This represents and forms a first antigen-binding site that binds to the CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above and / or in Table 4 are V H1 and V L1 This represents CD28 Polypeptide It forms a first antigen-binding site that binds to V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H3 and V L3 This forms a third antigen-binding site that binds to tumor target proteins (including, but not limited to, CD38 or HER2).

[0174] Exemplary anti-CD28 antigen-binding site sequences are provided in Table 4. In some embodiments, the anti-CD28 antigen-binding site of this disclosure comprises all of the CDR sequences 1, 2, 3, 4, 5, or 6 of the anti-CD28 antibody listed in Table 4. In some embodiments, the anti-CD28 antigen-binding site of this disclosure comprises the VH domain sequence and / or VL domain sequence of the anti-CD28 antibody listed in Table 4.

[0175] [Table 6]

[0176] Anti-CD3 binding site Certain aspects of this disclosure relate to binding proteins containing antigen-binding sites that bind to CD3 polypeptides. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, such as CD3-delta (also known as T3D, IMD19, and CD3-delta), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-gamma). Human CD3 polypeptides are known in the art, but are not limited to those represented by NCBI accession numbers XP_006510029.1 or NP_031674.1, or polypeptides produced from NCBI gene identification numbers 915, 916, or 917. In some embodiments, the binding protein containing an antigen-binding site that binds to a CD3 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, and It is multispecific and / or polyvalent. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein comprising four polypeptides that form three antigen-binding sites. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide, and one of which binds to the CD38 polypeptide. In some embodiments, the binding protein containing an antigen-binding site that binds to the CD3 polypeptide is a triple-specific binding protein comprising four polypeptides that form three antigen-binding sites, one of which binds to the CD28 polypeptide, one of which binds to the CD3 polypeptide, and one of which binds to the HER2 polypeptide.

[0177] In some embodiments, the binding site to CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). L2 The CDR-L1 sequence of the domain contains an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61), and QSLVHENLRTY (SEQ ID NO: 62).

[0178] In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0179] In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0180] In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0181] In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence containing the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0182] In some embodiments, the binding site that binds to CD3 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQ The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of YPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and / or an antibody light chain variable (VL) domain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0183] In some embodiments, the binding site that binds to CD3 is An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95). The binding site includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site that binds to CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 95.

[0184] In some embodiments, the binding site that binds to CD3 is An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96). In some embodiments, the CD3-binding site contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 96. In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 96.

[0185] In some embodiments, the binding site that binds to CD3 is An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97). In some embodiments, the CD3-binding site contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 97. In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and an amino acid sequence of SEQ ID NO: 97. Contains antibody light chain variable (VL) domains.

[0186] In some embodiments, the binding site that binds to CD3 is An antibody heavy chain variable (VH) domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the CD3-binding site contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 98.

[0187] In some embodiments, the binding site that binds to CD3 is A variable (VH) antibody heavy chain domain containing an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302) and / or The antibody light chain variable (VL) domain contains an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95). In some embodiments, the CD3-binding site contains an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 302 and / or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site for CD3 includes an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 302, and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 95.

[0188] In some embodiments of the above embodiments, the binding protein is a triple-specific binding protein. In some embodiments, the triple-specific binding protein is a tumor The binding protein includes an antigen-binding site that binds to a target antigen protein (but not limited to CD38 or HER2), an antigen-binding site that binds to a CD28 polypeptide, and an antigen-binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides, each containing three antigen-binding sites, where the polypeptide of formula I and the polypeptide of formula II form a crossover light-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the above-described anti-CD3 antigen-binding sites are V H2 and V L2 This represents a second antigen-binding site that binds to the CD3 polypeptide. In some embodiments, V H1 and V L1 The VH and VL domains of either of the anti-CD3 antigen-binding sites described above and / or in Table 5, which form the first antigen-binding site that binds to the CD28 polypeptide, are V H2 and V L2 It represents a second antigen-binding site that binds to the CD3 polypeptide, V H3 and V L3 This forms a third antigen-binding site that binds to tumor target proteins (including, but not limited to, CD38 or HER2).

[0189] Exemplary anti-CD3 antigen-binding site sequences are provided in Table 5. In some embodiments, the anti-CD3 antigen-binding site of this disclosure comprises all 1, 2, 3, 4, 5, or 6 CDR sequences of the anti-CD3 antibody listed in Table 5. In some embodiments, the anti-CD3 antigen-binding site of this disclosure comprises the VH domain sequence and / or VL domain sequence of the anti-CD3 antibody listed in Table 5.

[0190] [Table 7] [Table 8]

[0191] Linker In some embodiments, linkers L1, L2, L3, and L4 are in the range of no amino acids (length = 0) to about 100 amino acids in length, or less than 100, less than 50, less than 40, less than 30, less than 20, or less than 15 amino acids, or fewer. Linkers may be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. L1, L2, L3, and L4 in a single binding protein may all have the same amino acid sequence, or they may all have different amino acid sequences.

[0192] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71), and DKTHT (SEQ ID NO: 66), as well as those disclosed in International Publications WO2017 / 074878 and WO2017 / 180913. The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers containing randomly selected amino acids from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable in binding proteins.

[0193] The identity and sequence of amino acid residues in a linker can be modified depending on the type of secondary structural element that needs to be achieved in the linker. For example, glycine, serine, and alanine are best for linkers with the greatest flexibility. If a stiffer and longer linker is required, some combinations of glycine, proline, threonine, and serine are useful. Any amino acid residue can be considered as a linker in combination with other amino acid residues to construct the larger peptide linker required depending on the desired properties.

[0194] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, L1 is the length of 3 to 12 amino acid residues, L2 is the length of 3 to 14 amino acid residues, L3 is the length of 1 to 8 amino acid residues, and L4 is the length of 1 to 3 amino acid residues. In some embodiments, L1 is the length of 5 to 10 amino acid residues, L2 is the length of 5 to 8 amino acid residues, L3 is the length of 1 to 5 amino acid residues, and L4 is the length of 1 to 2 amino acid residues. In some embodiments, L1 is the length of 7 amino acid residues, L2 is the length of 5 amino acid residues, L3 is the length of 1 amino acid residue, and L4 is the length of 2 amino acid residues.

[0195] In some embodiments, L1, L2, L3, and L4 each independently contain a sequence that is zero amino acid in length or is selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1, L2, L3, and L4 each independently contain a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1 contains the sequence GQPKAAP (SEQ ID NO: 67), L2 contains the sequence TKGPS (SEQ ID NO: 68), L3 contains the sequence S, and L4 contains the sequence RT.

[0196] In some embodiments, at least one of L1, L2, L3, or L4 includes the sequence DKTHT (sequence number 66). In some embodiments, L1, L2, L3, and L4 include the sequence DKTHT (sequence number 66).

[0197] Fc region and constant domain In some embodiments, the binding protein of this disclosure is C H1 It further comprises a second polypeptide chain including an Fc region linked to the immunoglobulin hinge region and C H2 and C H3 It contains an immunoglobulin heavy chain constant domain. In some embodiments, the binding protein of this disclosure is C H1 It further comprises a third polypeptide chain including an Fc region linked to the immunoglobulin hinge region and C H2 and C H3 It contains an immunoglobulin heavy chain constant domain. In some embodiments, the binding protein of this disclosure is C H1 A second polypeptide chain further comprising an Fc region linked to the hinge region and C of an immunoglobulin. H2 and C H3 A second polypeptide chain containing the constant domain of the immunoglobulin heavy chain, and C H1 A third polypeptide chain further comprising an Fc region linked to the hinge region and C of an immunoglobulin. H2 and C H3 It contains a third polypeptide chain, which includes an immunoglobulin heavy chain constant domain.

[0198] In some embodiments, the binding protein of the Disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is an antibody hinge, C H1 , C H2 , C H3 , and optionally C H4Includes a domain. In some embodiments, the Fc region is the Fc region of human IgG1. In some embodiments, the Fc region is the Fc region of human IgG4. In some embodiments, the Fc region includes one or more of the mutations described below. In some embodiments, the Fc region is one Fc region of the binding protein heavy chain polypeptide (e.g., polypeptide 2 or 3) shown in Table 4. In some embodiments In this embodiment, the heavy chain constant region is one constant region of the heavy chain polypeptide of the binding protein shown in Table 4 (e.g., polypeptide 2 or 3). In some embodiments, the light chain constant region is one constant region of the light chain polypeptide of the binding protein shown in Table 4 (e.g., polypeptide 1 or 4).

[0199] In some embodiments, the binding proteins of this disclosure comprise one or two Fc variants. The term “Fc variant,” as used herein, refers to a molecule or sequence that has been modified from native Fc but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Therefore, the term “Fc variant” may include molecules or sequences that have been humanized from non-human native Fc. Furthermore, native Fc may include regions that can be removed to provide structural features or biological activity not required for the antibody-like binding proteins of the present invention. Therefore, the term “Fc variant” includes molecules or sequences that lack one or more native Fc sites or residues, or have one or more Fc sites or residues modified, that affect or are involved in (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity in expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC).

[0200] In some embodiments, the binding protein of this disclosure (e.g., a trispecific binding protein) includes a “knob” mutation in the second polypeptide chain and a “hole” mutation in the third polypeptide chain. In some embodiments, the binding protein of this disclosure includes a “knob” mutation in the third polypeptide chain and a “hole” mutation in the second polypeptide chain. In some embodiments, the “knob” mutation includes a substitution at positions corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the “knob” mutation includes a substitution at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the “whole” mutation includes substitutions at positions 407 of human IgG1 or IgG4 according to the EU index, and optionally at positions 349, 366, and / or 368. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and / or L368A. In some embodiments, the “whole” mutation includes substitutions at positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0201] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W or T366Y and optionally S354C; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to position 407 and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being Y40 7V or Y407T, and optionally Y349C, T366S, and / or L368A.

[0202] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising the hinge region of the immunoglobulin and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to position 407 of human IgG1 or IgG4 and optionally positions 349, 366, and / or 368 according to the EU index, the amino acid substitutions being Y407V or Y407T and optionally Y349 The amino acid substitutions are C, T366S, and / or L368A; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising the hinge region of the immunoglobulin and the constant domains of the CH2 and CH3 immunoglobulin heavy chains, the second Fc region comprising amino acid substitutions at positions corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being T366W or T366Y and optionally S354C.

[0203] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to positions 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V.

[0204] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising an amino acid substitution at a position corresponding to positions 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W.

[0205] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the hinge region of the immunoglobulin and the CH2 and CH3 immunoglobulins. The polypeptide chain comprises a globulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising the hinge region of the immunoglobulin and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. In some embodiments, the first and / or second Fc region is the Fc region of human IgG1. In some embodiments, the first and / or second Fc region is the Fc region of human IgG4.

[0206] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K; the third polyp The lipeptide chain further comprises a second Fc region linked to CH1, which is the Fc region of human IgG4 containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, and the second Fc region contains amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y4 The amino acids are 07V and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K.

[0207] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W; the third polyp The lipeptide chain further comprises a second Fc region linked to CH1, the second Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains The first Fc region contains amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V; the third polypeptide chain further contains a second Fc region linked to CH1, the second Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region containing amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W.

[0208] In some embodiments, the binding proteins of this disclosure include one or more mutations that can reduce effector functions, such as antibody-dependent cell phagocytosis (ADCP), complement-dependent cell damage (CDC), and / or antibody-dependent cell damage (ADCC) mediated by the Fc receptor. In some embodiments, the second polypeptide chain is C H1 It further includes a first Fc region linked to the first Fc region, the hinge region of the immunoglobulin and C H2 and C H3 It contains an immunoglobulin heavy chain constant domain; the third polypeptide chain is C H1 It further includes a second Fc region linked to the immunoglobulin hinge region and C H2 and C H3 The immunoglobulin heavy chain comprises a constant domain; the first and second Fc regions are the Fc regions of human IgG1; the first and second Fc regions each contain amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the second polypeptide chain comprises C H1 It further includes a first Fc region linked to the first Fc region, the hinge region of the immunoglobulin and C H2 and C H3 It contains an immunoglobulin heavy chain constant domain; the third polypeptide chain is C H1 It further includes a second Fc region linked to the immunoglobulin hinge region and C H2 and C H3The immunoglobulin heavy chain comprises a constant domain; the first and second Fc regions are the Fc regions of human IgG1; the first and second Fc regions each contain amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the second and third polypeptide chains also contain Fc regions of human IgG1, each containing amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are the Fc regions of human IgG4, and each Fc region contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. In some embodiments, the binding protein is C H1 A second polypeptide chain further comprising a first Fc region linked to the first Fc region, wherein the first Fc region is the hinge region and C of the immunoglobulin. H2 and C H3 A second polypeptide chain containing the constant domain of the immunoglobulin heavy chain, and C H1 A third polypeptide chain further comprising a second Fc region linked to the hinge region and C of an immunoglobulin. H2 and C H3 It comprises a third polypeptide chain containing an immunoglobulin heavy chain constant domain; the first and second Fc regions each contain amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

[0209] In some embodiments, the second polypeptide chain is a first Fc region linked to CH1. The region further includes the first Fc region, which is the Fc region of human IgG4 containing the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, and the first Fc region contains amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; the third polypeptide chain is the second linked to CH1 The Fc region further comprises the Fc region of human IgG4, which includes the hinge region of the immunoglobulin and the constant domains of the CH2 and CH3 immunoglobulin heavy chains, and the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y4 The amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being the Fc region of human IgG4 including the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K.

[0210] In some embodiments, the Fc region is the Fc region of human IgG4 containing one or more mutations that reduce or eliminate the binding of FcγI and / or FcγII. In some embodiments, the Fc region is the Fc region of human IgG4 containing one or more mutations that reduce or eliminate the binding of FcγI and / or FcγII but do not affect the binding of FcRn. In some embodiments, the Fc region is the Fc region of human IgG4 containing an amino acid substitution at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is the Fc region of human IgG4 containing an amino acid substitution at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is the Fc region of human IgG4 containing amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is the Fc region of human IgG4 containing amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and deletions at 236. In some embodiments, the Fc region is the Fc region of human IgG4 containing amino acid mutations at positions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are deletions at S228P;E233P, F234V, L235A, and 236; and / or R409K.

[0211] In some embodiments, the Fc region is used for Fc receptor binding and / or Ef. This includes one or more mutations that reduce or eliminate receptor function (e.g., antibody-dependent cell phagocytosis (ADCP), complement-dependent cell damage (CDC), and / or antibody-dependent cell damage (ADCC) mediated by the Fc receptor).

[0212] In some embodiments, the Fc region is the Fc region of human IgG1 containing one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is the Fc region of human IgG1 containing amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0213] In some embodiments, the binding protein of this disclosure includes one or more mutations to improve stability, e.g., the stability of the hinge region and / or dimer boundary of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583~26593). In some embodiments, the mutations include substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. In some embodiments, the binding protein is C H1 A second polypeptide chain further comprising a first Fc region linked to the first Fc region, wherein the first Fc region is the hinge region and C of the immunoglobulin. H2 and C H3 A second polypeptide chain containing the constant domain of the immunoglobulin heavy chain, and C H1 A third polypeptide chain further comprising a second Fc region linked to the hinge region and C of an immunoglobulin. H2 and C H3The present disclosure comprises a third polypeptide chain containing an immunoglobulin heavy chain constant domain; the first and second Fc regions are the Fc regions of human IgG4; and the first and second Fc regions each contain amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. In some embodiments, the binding protein of this disclosure includes knob and hole mutations as well as one or more mutations to improve stability. In some embodiments, the first and / or second Fc regions are the Fc regions of human IgG4.

[0214] In some embodiments, the Fc region is the Fc region of human IgG1 containing one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is the Fc region of human IgG1 containing amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0215] nucleic acid Other aspects of this disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any binding protein described herein. Exemplary and non-exclusive nucleic acid sequences are provided in Table 5.

[0216] Other aspects of the present disclosure relate to a polynucleotide kit, wherein the polynucleotide encodes one or more polypeptides of binding proteins, such as those described herein. In some embodiments, the polynucleotide kit of the present disclosure comprises (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189 (b) a polynucleotide containing the polynucleotide sequence of SEQ ID NO: 190, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 200; (d) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 201, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 202, and the polynucleotide sequence of SEQ ID NO: 203 (e) a third polynucleotide containing the sequence, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 204; (f) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 205, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 206, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 207, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 208; (g) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 209, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 210, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 211, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 212; (g) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 213, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 214, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 215, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 216;(h) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 217, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 218, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 219, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 220; (i) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 221, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 222, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 223, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 224; (j) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 225, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 226, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 227, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 228; (k) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 229, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 230, (l) a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 231, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 232; (m) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 233, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 234, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 235, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 236; (m) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 237, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 238, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 239, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 240; (n) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 241, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 242, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 243, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 244;(o) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 245, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 246, and a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 247; (p) a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 248; (p) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 249, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 250, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 251, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 252; (q) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 253, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 254, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 255, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 256; (r) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 257, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 258, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 259, and the polynucleotide sequence of SEQ ID NO: 260 (s) A fourth polynucleotide containing the nucleotide sequence of SEQ ID NO: 261; (t) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 262; a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 263; a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 264; (u) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 265; a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 266; a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 267; and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 268; (u) A first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 269; a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 270; a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 271; and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 272;Or (v) a polynucleotide kit containing one, two, three, or four polynucleotides, including a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 273, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 274, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 275, and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 276.

[0217] Other aspects of this disclosure relate to vector systems comprising one or more vectors encoding first, second, third, and fourth polypeptide chains of any binding protein described herein. In some embodiments, the vector system comprises, for example, a first vector encoding a first polypeptide chain of the binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein, as shown in the polynucleotides of Table 6. In some embodiments, the vector system comprises a first vector encoding first and second polypeptide chains of the binding protein, and a second vector encoding third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding first and third polypeptide chains of the binding protein, and a second vector encoding second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding first and fourth polypeptide chains of the binding protein, and a second vector encoding second and third polypeptide chains of the binding protein. In some embodiments, the vector system includes a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. One or more vectors in the vector system may be any vector described herein. In some embodiments, one or more vectors are expression vectors. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, for example, 1, 2, 3, or 4 expression vectors.

[0218] Standard recombinant DNA methodologies are used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be carried out as commonly achieved in the art or as described herein, according to the manufacturer's specifications. Unless otherwise specified, the academic terms used herein in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Similarly, prior art can be used for chemical synthesis, chemical analysis, pharmaceutical manufacturing, formulation, delivery, and patient treatment.

[0219] In some embodiments, isolated nucleic acids are operably ligated to a heterologous promoter to direct the transcription of a binding protein-coding nucleic acid sequence. The promoter may refer to a nucleic acid regulatory sequence that directs the transcription of the nucleic acid. The first nucleic acid sequence is operably ligated to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, the promoter is operably ligated to the binding protein-coding sequence when the promoter affects the transcription or expression of the coding sequence. Examples of promoters include, but are not limited to, those derived from the genomes of viruses (e.g., polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian virus 40 (SV40)), heterologous eukaryotic promoters (e.g., actin promoter, immunoglobulin promoter, and heat shock promoter), CAG promoters (Niwa et al., Gene 108(2):193-199, 1991), phosphoglycerate kinase (PGK) promoters, tetracycline-inducible promoters (Masui et al., Nucleic Acid Res.33:e43, 2005), the lac system, trp system, tac system, trc system, major operator and promoter regions of phage lambda, the 3-phosphoglycerate kinase promoter, the yeast acid phosphatase promoter, and the yeast alpha-conjugation factor promoter. The polynucleotides encoding the binding proteins of this disclosure may be under the control of a constitutive promoter, an inductive promoter, or any other suitable promoter described herein or any other suitable promoter readily recognizable by those skilled in the art.

[0220] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably ligated to the polynucleotide to be expressed. The term “regulatory sequence” includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic viruses (e.g., SV40 enhancers on the late side (bp 100-270) of the origin of replication, cytomegalovirus early promoter enhancers, polyoma enhancers on the late side of the origin of replication, adenovirus enhancers, etc.). Suitable vector examples include plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbisvirus, measles virus, herpesvirus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells. Yes, it is possible. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cells of interest.

[0221] host cell Other aspects of the present disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. In some embodiments, the isolated host cell of the present disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five® cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells include, for example, human fetal kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293® cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human hepatocytes (e.g., Hep G2, HB 8065), and mouse mammary gland tumor cells (e.g., MMT). Examples include 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma cells (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0222] Other aspects of this disclosure relate to methods for producing any binding protein described herein. In some embodiments, the method comprises: a) culturing a host cell (e.g., any host cell described herein) containing an isolated nucleic acid, vector, and / or vector system (e.g., any isolated nucleic acid, vector, and / or vector system described herein) under conditions such that the host cell expresses a binding protein; and b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions for protein expression are well known to those skilled in the art. Methods for isolating proteins from cultured host cells are also well known to those skilled in the art, and include, for example, affinity chromatography (e.g., two-step affinity chromatography including protein A affinity chromatography followed by size exclusion chromatography).

[0223] Pharmaceutical composition Therapeutic or pharmaceutical compositions containing binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions may contain a therapeutically effective amount of binding protein, or a binding protein-drug conjugate, in combination with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0224] Acceptable formulation materials are non-toxic to the recipient at the dosage and concentration used.

[0225] The pharmaceutical composition may contain, for example, formulation materials for modifying, maintaining, or preserving the composition's pH, volumetric osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeation.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), and preservatives (e.g., benzalkonium chloride, ammonium compounds). This includes, but is not limited to, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, e.g., sodium or potassium chloride, or mannitol, sorbitol), delivery media, diluents, excipients, and / or pharmaceutically adjuvants (e.g., incorporated herein by reference for any purpose, REMINGTON'S See PHARMACEUTICAL SCIENCES (18th Ed., edited by AR Gennaro, Mack Publishing Company 1990) and subsequent editions thereof.

[0226] The optimal pharmaceutical composition can be determined by those skilled in the art, for example, depending on the intended route of administration, delivery format, and desired dosage. Such a composition can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0227] The primary medium or carrier in a pharmaceutical composition may be either aqueous or non-aqueous. For example, suitable mediums or carriers for injection may be water, saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary mediums. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of this disclosure, a binding protein composition can be prepared for storage by mixing a selected composition having the desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution. Furthermore, the binding protein can be formulated as a lyophilized product using a suitable excipient, such as sucrose.

[0228] The pharmaceutical compositions of this disclosure may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the gastrointestinal tract, for example, orally. The manufacture of such pharmaceutically acceptable compositions is within the technical scope of the art.

[0229] The formulation components are present at concentrations acceptable to the administration site. For example, buffers are used to maintain the composition within a pH range of physiological pH or slightly lower, typically around 5 to 8.

[0230] When parenteral administration is intended, the therapeutic composition for use may be in the form of a pyrogenically free, parenterally acceptable, aqueous solution containing the desired binding protein in a pharmaceutically acceptable medium. A particularly preferred medium for parenteral injection is sterile distilled water, in which the binding protein is formulated as a properly preserved sterile isotonic solution. Yet another product may involve formulation of the desired molecule using agents that provide controlled or sustained release of the product which can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymer compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes. Hyaluronic acid may also be used, which may have the effect of promoting a sustained duration of action in circulation. Other preferred means for introducing the desired molecule include implantable drug delivery devices.

[0231] In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated using a propellant for aerosol delivery. In yet another embodiment, the solution can be sprayed.

[0232] It is also intended that certain formulations may be administered orally. In one embodiment of this disclosure, the binding protein administered in this manner may be formulated with or without carriers conventionally used in solid-state formulations, such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to promote the absorption of the binding protein. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0233] Another pharmaceutical composition may involve an effective amount of binding protein in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0234] Additional pharmaceutical compositions of this disclosure will be apparent to those skilled in the art and include formulations involving binding proteins in sustained or controlled delivery formulations. Various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art. Additional examples of sustained-release products include semipermeable polymer matrices in the form of molded articles, such as films, or microcapsules. The sustained-release matrix may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyrate. The sustained-release compositions may also include liposomes, which can be produced by any of several methods known in the art.

[0235] Pharmaceutical compositions used for in vivo administration must typically be sterile. This can be achieved by filtration through a sterile filtration membrane. The composition is lyophilized. If applicable, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Additionally, parenteral compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be penetrated by subcutaneous needles.

[0236] Once a pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as an anhydrous or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form requiring reconstitution before administration (e.g., a lyophilized form).

[0237] This disclosure also includes kits for generating single-dose units. Each kit may contain both a first container containing a dry protein and a second container containing an aqueous formulation. Kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of this disclosure.

[0238] The effective amount of a therapeutically used binding protein pharmaceutical composition depends, for example, on the therapeutic context and purpose. Those skilled in the art will understand that the appropriate dosage level for a treatment will therefore vary, in part, on the molecules being delivered, the indications for which the binding protein is used, the route of administration, and the patient's size (body weight, body surface area, or organ size) and condition (age and overall health). Thus, clinicians can titrate the dosage and modify the route of administratio...

Claims

1. The use of a binding protein in the manufacture of a drug for expanding and proliferating T cells in a patient, wherein the binding protein is a binding protein comprising four polypeptide chains that form three antigen-binding sites, The first polypeptide chain is given by formula: V L2 -L 1 -V L1 -L 2 -C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L 3 -V H2 -L 4 -C H1 -Hinge - C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 Domain and C H2 It is an immunoglobulin hinge region that connects domains; L 1 , L 2 , L 3 and L 4 It is an amino acid linker; Polypeptides of formula I and formula II form crossover light-heavy chain pairs; V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide; V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), V L2 The domain includes a CDR-L1 sequence containing an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61), and QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); V H3 and V L3 It forms a third antigen-binding site that binds to tumor target proteins. The aforementioned use.

2. The use according to claim 1, wherein the T cells express a chimeric antigen receptor (CAR) on their cell surface or contain a polynucleotide encoding a CAR.

3. The use according to claim 1 or 2, wherein the T cells are memory T cells or effector T cells.

4. The use according to claim 3, wherein the memory T cells are CD8+ or CD4+ memory T cells.

5. The aforementioned memory T cells are central memory T cells (T CM ) or effector memory T cells (T EM The use according to claim 3 or 4, which is the same as the use described in claim 3 or 4.

6. The aforementioned V H1 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and a CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51), V L1 The use according to any one of claims 1 to 5, wherein the domain comprises a CDR-L1 sequence containing the amino acid sequence of QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53), and a CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (SEQ ID NO: 54).

7. The aforementioned V H1 The domain is The amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTTSISTAYMELSRLRSDDTAVYYCTRSHHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) is included and / or V L1 The domain is The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAAPKLLLIYKASNLHTGVPPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92) is included. The use described in claim 6.

8. The aforementioned V H2 The domain is QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYAT YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVS S (Sequence ID 93), or The amino acid sequence includes QVQLVESGGGGVVQPGRSLRLSCAASGFTFFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302), and / or the V L2 The domain is, DIVMTQTPPLLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFFSGSGSGTDFTTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 95), DIVMTQTPPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFFSGSGSGTDFTTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 96), DIVMTQTPPLSLSVTPGQPASISCKSSQSLLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFFSGSGSGTDFTTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 97), and The amino acid sequence includes one selected from the group consisting of DIVMTQTPPLLSLSVTPGQPASISCKSSQSLLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGSGTDFTTLKISRVEEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (Sequence ID 98), The use according to any one of claims 1 to 7.

9. The use according to any one of claims 1 to 8, wherein the tumor target protein is a CD38 polypeptide.

10. The use according to claim 9, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide.

11. (a) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QSVSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18); (b) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence containing the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24); (c) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR-H2 sequence containing the amino acid sequence of INPGSGST (SEQ ID NO: 26), and a CDR-H3 sequence containing the amino acid sequence of ARYAYGY (SEQ ID NO: 27), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QNVGTA (SEQ ID NO: 28), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 29), and a CDR-L3 sequence containing the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30); (d) V H3 The domain contains the amino acid sequence GYSFTNYA (SEQ ID NO: 31) on a CD. It includes the R-H1 sequence, the CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and the CDR-H3 sequence containing the amino acid sequence of ARRFEGYYSMDY (SEQ ID NO: 33), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36); (e) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence containing the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42); or (f) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence containing the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45), V L3 The use according to claim 9, wherein the domain comprises a CDR-L1 sequence containing the amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48).

12. (a) V H3 The domain is The amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRRSEDTAVYFCARTGGGLRRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79) contains and / or V L3 The domain is The amino acid sequence DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80) is included; (b) V H3 The domain is The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGGFPDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRRFFDWFWGQGTLVTVSS (SEQ ID NO: 81) is included and / or V L3 The domain is The amino acid sequence contains EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAAPRRLLIYGASTRATGIPVRFSGSGSGTTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82); (c) V H3 The domain is The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYAAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTTMTVDRSSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83) contains and / or V L3 The domain is The amino acid sequence contains DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (Sequence ID 84); (d) V H3 The domain is QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQA The amino acid sequence contains PGQGLEWMGVISPYYGDTTYAQKFQGRVTTMTVDKSSSSTAYMELSRLRSDDDTAVYYCARRFEGYYSMDYWGQGTLVTVSS (SEQ ID NO: 85), and / or V L3 The domain is The amino acid sequence DVVMTQSPLSLLPVTLGQPASISCRPSQSLLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGGSGSGTDFTTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (Sequence ID 86) is included; (e) V H3 The domain is The amino acid sequence QVQLVESGGGGVVQPGRSLRLSCAASGFTFFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87) is included and / or V L3 The domain is The amino acid sequence contains DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAAPKLLIFAASTLHSGVPSRFSGSGSGTEFFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88); (f) V H3 The domain is The amino acid sequence QVQLVESGGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89) contains and / or V L3 The domain is The amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLLIYAASSLQSGVPPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90) is included; (g) V H3 The domain is The amino acid sequence QVQLQQSGPELVRPGTSVKVSCKASGYAAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTTLTVSS (SEQ ID NO: 277) is included and / or V L3 The domain is Containing the amino acid sequence DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO: 278); or (h)V H3 The domain contains the amino acid sequence QVQLLQSGAELVRRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSSTAYMELARLTSEDSAIYFCCARRFEGYYYSMDYWGQGTSVTVSS (SEQ ID NO: 279), and / or V L3 The domain is The amino acid sequence of DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGGSGSGTDFFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK (Sequence ID 280) is included. The use described in claim 11.

13. (a) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence identical to that of SEQ ID NO: 156 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence identical to that of SEQ ID NO: 157 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence identical to that of SEQ ID NO: 158 by at least 95% The amino acid sequence is included; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159; (b) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence identical to that of SEQ ID NO: 160 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence identical to that of SEQ ID NO: 161 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence identical to that of SEQ ID NO: 162 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence identical to that of SEQ ID NO: 163 by at least 95%; (c) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 166; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 167; (d) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence identical to that of SEQ ID NO: 168 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence identical to that of SEQ ID NO: 169 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence identical to that of SEQ ID NO: 170 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence identical to that of SEQ ID NO: 171 by at least 95%; (e) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence identical to that of SEQ ID NO: 172 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence identical to that of SEQ ID NO: 173 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence identical to that of SEQ ID NO: 174 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence identical to that of SEQ ID NO: 175 by at least 95%; (f) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence identical to that of SEQ ID NO: 176 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence identical to that of SEQ ID NO: 177 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence identical to that of SEQ ID NO: 178 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence identical to that of SEQ ID NO: 179 by at least 95%; (g) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 183; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 184; or (h) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence identical to that of SEQ ID NO: 185 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence identical to that of SEQ ID NO: 186 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence identical to that of SEQ ID NO: 187 by at least 95% The use according to claim 9, comprising the amino acid sequence of; the fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO:

188.

14. The use according to any one of claims 1 to 8, wherein the tumor target protein is a HER2 polypeptide.

15. The use according to claim 14, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the HER2 polypeptide is a human HER2 polypeptide.

16. (a) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (b) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (c) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (d) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8). L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (e) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8), and the V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (f) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); or (g) V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence containing the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). The use described in claim 14.

17. (a) V H3 The domain is EVQLVESGGGLVQPGGSLRLSCASGFNIKDTYIHWVQRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGYYAMDYWGQGTVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCASGFNIRDTYIHWVQRQAPGKGLEWVARIYPTQGYYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCASGFNIRDTYIHWVQRQAPGKGLEWVARIYPTQGYYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGYYAMDYWGQGTVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCASGFNIRDTYIHWVQRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGYYAMDYWGQGTVTVSS (SEQ ID NO: 75), or EVQLVESGGGLVQPGGSLRLSCASGFNIRDTYIHWVQRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTVTVSS (SEQ ID NO: 76) amino acid sequence, and / or V L3 The domain contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAWAYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVTAVAYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTVEIK (SEQ ID NO: 78); (b) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) and / or V L3 The domain is The amino acid sequence contains DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 77); (c) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73) and / or V L3 The domain is The amino acid sequence contains DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 77); (d) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) and / or V L3 The domain is The amino acid sequence contains DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 77); (e) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74) and / or V L3 The domain is The amino acid sequence contains DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 77); (f) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76) and / or V L3 The domain is The amino acid sequence comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77); or (g) V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) and / or V L3 The domain is The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 78) is included. The use described in claim 16.

18. (a) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 102; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 103; (b) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence identical to that of SEQ ID NO: 104 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence identical to that of SEQ ID NO: 105 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence identical to that of SEQ ID NO: 106 by at least 95% The amino acid sequence is included; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107; (c) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence identical to that of SEQ ID NO: 112 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence identical to that of SEQ ID NO: 113 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence identical to that of SEQ ID NO: 114 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence identical to that of SEQ ID NO: 115 by at least 95%; (d) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 130; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 131; (e) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence identical to that of SEQ ID NO: 136 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence identical to that of SEQ ID NO: 137 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence identical to that of SEQ ID NO: 138 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence identical to that of SEQ ID NO: 139 by at least 95%; (f) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence identical to that of SEQ ID NO: 140 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence identical to that of SEQ ID NO: 141 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence identical to that of SEQ ID NO: 142 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence identical to that of SEQ ID NO: 143 by at least 95%; (g) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence identical to that of SEQ ID NO: 144 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence identical to that of SEQ ID NO: 145 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence identical to that of SEQ ID NO: 146 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence identical to that of SEQ ID NO: 147 by at least 95%; (h) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence identical to that of SEQ ID NO: 152 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence identical to that of SEQ ID NO: 153 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence identical to that of SEQ ID NO: 154 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence identical to that of SEQ ID NO: 155 by at least 95%; (i) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence identical to that of SEQ ID NO: 286 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence identical to that of SEQ ID NO: 287 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 288 or an amino acid sequence identical to that of SEQ ID NO: 288 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 289 or It contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 289; (j) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 290 or an amino acid sequence identical to that of SEQ ID NO: 290 by at least 95%; the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 291 or an amino acid sequence identical to that of SEQ ID NO: 291 by at least 95%; the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 292 or an amino acid sequence identical to that of SEQ ID NO: 292 by at least 95%; the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 293 or an amino acid sequence identical to that of SEQ ID NO: 293 by at least 95%; (k) The use according to claim 14, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 294 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 295 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 296 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO:

297.

19. The use of a binding protein in the manufacture of a drug for expanding and proliferating T cells in a patient, wherein the binding protein is a binding protein comprising four polypeptide chains that form three antigen-binding sites, The first polypeptide chain is given by formula: V L2 -L 1 -V L1 -L 2 -C L [I] It includes a structure represented by, The second polypeptide chain is given by formula: V H1 -L 3 -V H2 -L 4 -C H1 -Hinge-C H2 -C H3 [II] It includes a structure represented by, The third polypeptide chain is given by formula: V H3 -C H1 -Hinge-C H2 -C H3 [III] It includes a structure represented by, The fourth polypeptide chain is given by formula: V L3 -C L [IV] It includes a structure represented by, Here, V L1 This is the first immunoglobulin light chain variable domain; V L2 This is the second immunoglobulin light chain variable domain; V L3 This is the third immunoglobulin light chain variable domain; V H1 This is the first immunoglobulin heavy chain variable domain; V H2 This is the second immunoglobulin heavy chain variable domain; V H3 This is the third immunoglobulin heavy chain variable domain; C L This is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 It is a heavy chain constant domain; C H2 is immunoglobulin C H2 It is a heavy chain constant domain; C H3 is immunoglobulin C H3 It is a heavy chain constant domain; The hinge is C H1 Domain and C H2 It is an immunoglobulin hinge region that connects domains; L 1 , L 2 , L 3 and L 4 It is an amino acid linker; Polypeptides of formula I and formula II form crossover light-heavy chain pairs; The aforementioned V H1 and V L1 This forms a first antigen-binding site that binds to the CD28 polypeptide, where V H1 The domain contains the amino acid sequence GYTFTSYY (SEQ ID NO: 49) C It includes the DR-H1 sequence, the CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and the CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51), and V L1 The domain includes a CDR-L1 sequence containing the amino acid sequence QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence containing the amino acid sequence KAS (SEQ ID NO: 53), and a CDR-L3 sequence containing the amino acid sequence QQGQTYPY (SEQ ID NO: 54); The aforementioned V H2 and V L2 This forms a second antigen-binding site that binds to the CD3 polypeptide, and here, V H2 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and V L2 The domain includes a CDR-L1 sequence containing QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and The aforementioned V H3 and V L3 This forms a third antigen-binding site that binds to the HER2 polypeptide, where V H3 The domain includes a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence containing the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and V L3 The domain includes a CDR-L1 sequence containing the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12), as described above.

20. The use according to claim 19, The aforementioned V H1 The domain is QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) contains the amino acid sequence, and V L1 The domain is The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAAPKLLLIYKASNNLHTGVPPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92) is included; The aforementioned V H2 The domain is The amino acid sequence QVQLVESGGGGVVQPGRSLRLSCAASGFTFFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) is included, and V L2 The domain contains the amino acid sequence DIVMTQTPPLSLLSVTPGQPASISCKSSQSLLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFFSGSGSGTDFTTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95); and The aforementioned V H3 The domain is The amino acid sequence contains EVQLVESGGGLLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and V L3 The domain contains the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77), The aforementioned use.

21. The use according to claim 19, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO:

107.

22. Said L 1 , L 2 , L 3 and L 4 Each of these independently comprises the sequence DKTHT (sequence number 66), as described in any one of claims 19 to 21.

23. The hinge-C of the second and third polypeptide chains. H2 -C H3 The domain is human IgG4-hinge-C. H2 -C H3 This is a domain, and here the hinge-C H2 -C H3 The use according to any one of claims 19 to 22, wherein each domain comprises an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, and the amino acid substitutions are F234A and L235A.

24. The hinge-C of the second and third polypeptide chains. H2 -C H3 The domain is human IgG4-hinge-C. H2 -C H3 This is a domain, and here the hinge-C H2 -C H3 The use according to any one of claims 19 to 23, wherein each domain contains an amino acid substitution at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, and the amino acid substitutions are S228P and R409K.

25. Hinge-C of the second polypeptide chain H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; and the hinge-C of the third polypeptide chain. H2 -C H3 The domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. or use as described in any one of claims 19 to 24.

26. Hinge-C of the second polypeptide chain H2 -C H3 The domain includes amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; and the hinge-C of the third polypeptide chain. H2 -C H3 The use according to any one of claims 19 to 24, wherein the domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU Index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

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