TW202104261A - Trispecific binding proteins, methods, and uses thereof - Google Patents

Abstract

Provided herein are trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein and a second pair of polypeptides possess a single variable domain forming a single antigen binding site. In some embodiments, the binding proteins comprise a binding site that binds a CD28 polypeptide, a binding site that binds a CD3 polypeptide, and a binding site that binds a third polypeptide, such as a tumor target protein. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.

Description

Trispecific binding protein, method and use

This article relates to a trispecific and/or trivalent binding protein, which comprises four polypeptide chains forming three antigen binding sites that specifically bind to one or more target proteins, wherein the binding The first pair of polypeptides of the protein possess dual variable domains with staggered orientation. This document also relates to methods for preparing trispecific and/or trivalent binding proteins and the use of such binding proteins.

Biotherapeutics based on monoclonal antibodies have become an important approach for the development of new drugs. Monoclonal antibody technology provides specific targeting, precise signal transduction delivery and/or payload for specific cell populations, and provides long-lasting biological effects through its Fc function. Efforts in antibody engineering have been able to develop bispecific antibodies that combine the specificities of two monoclonal antibodies for various biological applications, thereby expanding the scope of antibody drug development. The newly discovered neutralizing antibodies with improved breadth and efficacy can provide more options for the development of biotherapeutics for the treatment of complex diseases such as cancer, arthritis, and/or inflammatory disorders.

Immuno-oncology is a promising and emerging therapeutic method for cancer disease management. The immune system is the first line of defense against the development and progression of cancer. There is now a lot of evidence that T cells can control tumor growth in the early and late stages of the disease and prolong the survival of cancer patients. However, T cells specific to tumors may be restricted in many ways, preventing them from controlling the disease.

As a part of human adaptive immunity, T cell immunity plays a key role in controlling viral infections and cancer. It may eliminate infected cells and malignant cells, leading to the elimination of viral infections or the cure of cancer. In chronic infectious diseases such as herpes virus infection (HSV, CMV, EBV, etc.), HIV and HBV, the virus establishes its persistence in the human body through a variety of mechanisms including immunosuppression, T cell exhaustion, and establishment of incubation period. However, virus infection usually induces virus antigen-specific immunity including antigen-specific CD8 T cells that can easily recognize infected cells for mediation by cytokine release or cytotoxic T cells (CTL). The killing process of the guide is used to control or kill.

Therefore, in vivo and/or ex vivo virus antigen-specific T cell activation and/or expansion can provide a therapeutic strategy against chronic viral infections.

All references cited in this text, including patent applications, patent publications and UniProtKB/Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference was specifically and individually indicated by reference Incorporated.

To meet these and other needs, this article provides trispecific binding proteins (eg, antibodies) that form three antigen binding sites. These binding proteins can specifically bind to one, two or three antigen targets or target proteins, such as CD28, CD3 and tumor target proteins. Some tumors show specific antigens. For example, HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, and also in gastric, ovarian, lung, and prostate cancer. The optimal activation of T cells requires two factors: 1. Antigen recognition and 2. Co-stimulation. In the case of using the trispecific HER2/CD28xCD3 trispecific binding protein 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 trispecific binding protein recruits T cells to the tumor via HER2, CD38, or a binding site that recognizes another tumor target protein, and activates the joined T cells via anti-CD3 and CD28. The resulting activation induces the killing potential of the immune cells against nearby tumor cells. In addition, it is described that the anti-CD3 binding site has high affinity binding to human CD3 polypeptide and removes potential production disadvantages (for example, deamidation sites).

This article further provides anti-CD38/CD28xCD3 trispecific antibodies, developed and evaluated their potential in activating T cells and subsequently proliferating and/or expanding antigen-specific T cells. These tri-specific Abs can effectively expand CD4 and CD8 effector and memory populations in vitro, including antigen-specific CD8 T central memory cells and effector memory cells. Specifically, the in vitro expansion of CMV, EBV, HIV-1, influenza-specific CD8 central memory cells and effector memory cells was demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibit novel characteristics by conjugating CD3/CD28/CD38 to provide signal transduction pathways that stimulate and expand T cells, which can provide treatment for chronic infectious diseases (such as HSV, CMV) , EBV, HIV-1 and HBV infection).

In order to meet these and other needs, this document provides binding proteins that bind to CD38 polypeptides (for example, human and cynomolgus CD38 polypeptides). The binding proteins include monospecific, bispecific, and bispecific antigen binding sites that bind to CD38 polypeptides. Sexual or trispecific binding protein. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, then activate T cells and promote the activated T cells to kill neighboring cancer cells through the granzyme/perforin mechanism, thereby providing an anti-CD38 antibody such as DARZALEX® (daratumumab) has different anti-tumor activity modes of action. In addition, the ability to bind both human and cyno CD38 polypeptides makes it easy to test the binding protein in preclinical toxicology studies, for example to evaluate its safety profile for future clinical applications.

In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third most It comprises a peptide chain represented by the structural formula: V _H3 -C _H1 - hinge -C _H2 -C _H3 [III] and a fourth polypeptide chain comprises a structure represented by the formula: V _L3 -C _L [IV] wherein: _VL1 is the variable domain of the first immunoglobulin light chain; _VL2 is the variable domain of the second immunoglobulin light chain; _VL3 is the variable domain of the third immunoglobulin light chain; 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; _CL is the immunoglobulin light chain constant structure domain; C C _{Hl Hl} immunoglobulin heavy chain constant domain; C C _{H2 of H2 of} immunoglobulin heavy chain constant domain; C _{H3 of} C _{H3 of} an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 hinge region of an immunoglobulin C _H2 domain; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said polypeptide of formula I and formula II of the light chain polypeptide form an interleaved -A heavy chain pair; and wherein V _H1 and V _L1 form a first antigen binding site; wherein V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, wherein the V _H2 domain comprises: containing GFTFTKAW ( The CDR-H1 sequence of the amino acid sequence of SEQ ID NO: 55), the CDR-H2 sequence of the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and the amino acid of RGVYYALSPFDY (SEQ ID NO: 57) CDR-H3 sequences, and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ is a or L and X ₃ is Q, QSLVHX R, or F ₁ NX ₂ X ₃ TY (SEQ ID NO: 180) CDR-L1 sequence of the amino acid sequence, CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and amino acid sequence containing GQGTQYPFT (SEQ ID NO: 65) CDR-L3 sequence; and where V _H3 and V _L3 form a third antigen binding site.

In some embodiments, the first binding site binds a CD28 polypeptide. In some embodiments, the V _H1 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50) -H2 sequence and CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51); and the V _L1 domain contains: CDR- containing the amino acid sequence of QNIYVW (SEQ ID NO: 52) L1 sequence, CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53), and CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). Amino acid sequence In some embodiments, the domain comprising V _H1 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO:: 91 ) amino acid sequences, and / or V _L1 domain comprises DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (92 SEQ ID NO) .

In some embodiments, the _{CDR-L1 sequence of the V L2} domain comprises 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 herein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain, which comprises an amino acid containing GFTFTKAW (SEQ ID NO: 55) The CDR-H1 sequence of the sequence, the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or The antibody light chain variable (VL) domain, which contains the CDR-L1 sequence containing the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), and the CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) L2 sequence and CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding protein herein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain, which comprises an amino acid containing GFTFTKAW (SEQ ID NO: 55) The CDR-H1 sequence of the sequence, the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or The antibody light chain variable (VL) domain comprising the CDR-L1 sequence containing the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), and the CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) L2 sequence and CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding protein herein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain, which comprises an amino acid containing GFTFTKAW (SEQ ID NO: 55) The CDR-H1 sequence of the sequence, the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or The antibody light chain variable (VL) domain comprising the CDR-L1 sequence containing the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), and the CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) L2 sequence and CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding protein herein comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain, which comprises an amino acid containing GFTFTKAW (SEQ ID NO: 55) The CDR-H1 sequence of the sequence, the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or The antibody light chain variable (VL) domain comprising the CDR-L1 sequence containing the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), and the CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) L2 sequence and CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the V _H2 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) amino acid sequences, and / or V _L2 domain comprises the amino acid sequence selected from the following: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the V _H2 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302) amino acid sequences, and / or V _L2 domain comprises selected the amino acid sequence: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the binding protein herein 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: 95. In some embodiments, the binding protein herein 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 herein 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 herein 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 herein 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: 98.

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 (for example, a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (for example, a human HER2 polypeptide). In some embodiments, tumor target proteins herein include, but are not limited to, 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 eosinophil chemokine), 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/eosinophil chemokine-2) , CCL25 (also known as TECK), CCL26 (also known as eosinophil chemokine-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, 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 (also known as ICOS), CD279 (also known as PD-1), 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, exonucleoside triphosphate diphosphate Phosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rβ, IL1, IL1A , IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL 9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor of IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 Catenin), ITK, KIR, LAG3, LAMP1, Leptin, LPFS2, Class II MHC, MUC-1, NCR3LG1, NKG2D, NTPD-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152 , SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), 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 of 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.

In some embodiments, the third antigen binding site binds to a human CD38 polypeptide. In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QSVSSYGQGF (16 SEQ ID NO): -H2 sequence containing ARTGGLRRAYFTY (15 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17) and CDR-L3 sequence containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QSVISRF (22 SEQ ID NO): -H2 sequence containing TTGRFFDWF (21 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23), and CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR containing the amino acid sequence of INPGSGST (SEQ ID NO: 26) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QNVGTA (28 SEQ ID NO): -H2 sequence containing ARYAYGY (27 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 29) and CDR-L3 sequence containing the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QSLVHSNGNTY (34 SEQ ID NO): -H2 sequence containing ARRFEGFYYSMDY (33 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 35), and CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36).

In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QGISSY (40 SEQ ID NO): -H2 sequence containing ARDPGLRYFDGGMDV (39 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QGIRND (46 SEQ ID NO): -H2 sequence containing ARMFRGAFDY (45 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47), and CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). Amino acid sequence In some embodiments, the V _H3 domain comprises QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:: 79 ) amino acid sequences, and / or the V _L3 domain comprises DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (80 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO:: 81 ) amino acid sequences, and / or the V _L3 domain comprises EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (82 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO:: 83 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (84 SEQ ID NO) . In some embodiments, the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87) The amino acid sequence and / or the V _L3 domain comprises DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88) amino acid sequence . In some embodiments, the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89) amino acid sequences, and / or the V _L3 domain comprises AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90) amino acid sequence . Amino acid sequence In some embodiments, the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO:: 85 ) amino acid sequences, and / or the V _L3 domain comprises DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (86 SEQ ID NO) .

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

In some embodiments, the third antigen binding site binds to a human HER2 polypeptide. In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), and a CDR-H1 sequence containing IYPTNGYT (SEQ ID NO: 3), the CDR-H2 sequence of the amino acid sequence of IYPTQGYT (SEQ ID NO: 4) or IYPTNAYT (SEQ ID NO: 5) and containing SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7) or SRWGGSGFYAMDY (SEQ ID NO: 8) CDR-H3 amino acid sequences of the sequence; V _L3 and said domain comprises: comprising QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10) the amino acid The CDR-L1 sequence of the sequence, the CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and the CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3) CDR- the amino acid sequence and domain comprises the V _L3;: (6 SEQ ID NO) of the amino acid sequence of a CDR-H3 sequence:: comprising QDVNTA (9 SEQ ID NO) -H2 sequence containing SRWGGDGFYAMDY L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QDVNTA (9 SEQ ID NO): -H2 sequence containing SRWGGEGFYAMDY (7 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QDVNTA (9 SEQ ID NO): -H2 sequence containing SRWGGSGFYAMDY (8 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QDVNTA (9 SEQ ID NO): -H2 sequence containing SRWGGSGFYAMDY (8 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5) CDR- the amino acid sequence and domain comprises the V _L3; CDR-H3 of the sequence of the amino acid sequence:: containing QDVNTA (9 SEQ ID NO): -H2 sequence containing SRWGGEGFYAMDY (7 SEQ ID NO) L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR containing the amino acid sequence of IYPTNGYT (SEQ ID NO: 3) CDR- the amino acid sequence and domain comprises the V _L3;: (6 SEQ ID NO) of the amino acid sequence of a CDR-H3 sequence:: comprising QDVQTA (10 SEQ ID NO) -H2 sequence containing SRWGGDGFYAMDY L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) , EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78) the amino acid sequence. Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 72 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (77 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 73 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (77 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 75 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (77 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 74 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (77 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 76 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (77 SEQ ID NO) . Amino acid sequence In some embodiments, the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:: 72 ) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (78 SEQ ID NO) .

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

In some embodiments that can be combined with any of the other embodiments described herein _{, at least one of L 1} , L ₂ , L ₃ or L ₄ is independently 0 amino acids in length. In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ are each independently the length of zero amino acids or comprise a sequence selected from: 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, L ₁ , L ₂ , L ₃ and L ₄ each independently comprise a sequence selected from: 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, L ₁ comprises the sequence GQPKAAP (SEQ ID NO: 67), L ₂ comprises the sequence TKGPS (SEQ ID NO: 68), L ₃ comprises the sequence S, and L ₄ comprises the sequence RT. In some embodiments, _{at least one of L 1} , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO: 66). In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ comprise the sequence DKTHT (SEQ ID NO: 66).

In some embodiments of any of embodiments may be in combination with other embodiments described herein, the second and the third hinge of the polypeptide chain -C _H2 -C _H3 domain of human IgG4 hinge -C _H2 - C _H3 domain, and wherein said hinge -C _H2 -C _H3 domains each comprise a substitution at position 234 according to the amino acid positions 235 and corresponding to the EU index of human IgG4, wherein said amino acid substitution is F234A and L235A. In some embodiments, the hinge -C said second and said third polypeptide chain _H2 -C _H3 domain of human IgG4 hinge -C _H2 -C _H3 domain, and wherein the hinge -C _{The H2- CH3} domains each comprise an amino acid substitution at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A, and the deletion at 236. In some embodiments, the hinge -C said second and said third polypeptide chain _H2 -C _H3 domain of human IgG4 hinge -C _H2 -C _H3 domain, and wherein the hinge -C _{The H2- CH3} domains each contain amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge -C said second and said third polypeptide chain _H2 -C _H3 domain is a human IgG1 hinge -C _H2 -C _H3 domain, and wherein the hinge -C _{The H2-} C _H3 domains each contain amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge -C said second and said third polypeptide chain _H2 -C _H3 domain is a human IgG1 hinge -C _H2 -C _H3 domain, and wherein the hinge -C _H2 -C _H3 domain comprises a substitution at each amino acid at a position corresponding to position 298, 299 and 300 of human IgG1 based on the EU index, wherein said amino acid substitution is S298N, T299A and Y300S. In some embodiments, the second hinge -C _H2 -C _H3 polypeptide chain domain comprises the amine according to the EU index at a position corresponding to the human IgG1 or IgG4, 349,366,368 and 407 acid substitutions, wherein said amino acid substitution is Y349C, T366S, L368A and Y407V; and the hinge -C chain wherein the third polypeptide domain comprises _H2 -C _H3 according to the EU index corresponding to the human IgG1 Or the amino acid substitutions at positions 354 and 366 of IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the second hinge -C _H2 -C _H3 polypeptide chain domain comprises amino acid substitution according to the EU index at a position corresponding to the human IgG1 or IgG4, 354 and 366, wherein said amino acid substitution is a T366W S354C and; and wherein the said third hinge -C _H2 -C _H3 polypeptide chain domain comprises at EU index positions 349,366 corresponding to human IgG1 or IgG4 according The amino acid substitutions at positions 368 and 407, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

In some embodiments, provided herein is an isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of any of the above embodiments. In some embodiments, provided herein is a performance vector comprising the nucleic acid molecule of any of the above embodiments. In some embodiments, provided herein is an isolated host cell comprising the nucleic acid molecule of any of the above embodiments or the expression vector of any of the above embodiments. In some embodiments, the host cell is a mammalian cell or an insect cell.

In some embodiments, provided herein is a pharmaceutical composition comprising the binding protein of any of the above embodiments and a pharmaceutically acceptable carrier.

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

In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the patient is a human.

In some embodiments, the third antigen binding site binds to a human CD38 polypeptide, and wherein cancer cells from the 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, before administering the binding protein, the patient has been treated with darlimumab without a washout period.

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

In some embodiments, provided herein is a method for amplifying virus-specific memory T cells, which comprises contacting the virus-specific memory T cells with a binding protein, wherein the binding protein comprises four polypeptides forming three antigen binding sites Chain, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1- L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain includes a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [ III] and the fourth polypeptide chain comprises the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the second immunoglobulin light chain 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 a third immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; an immunoglobulin C _H2 C _H2 heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ is an amino acid linker; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide, Wherein V _H2 and V _L2 form a second antigen binding site that binds to the CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and containing IKDKSNSYAT ( CDR-H2 sequence 56) and comprising the amino acid sequence RGVYYALSPFDY (SEQ ID NO:: SEQ ID NO CDR-H3 sequence of 57 amino acid sequence), and the V _L2 domain comprises: wherein X ₁ comprising is E or Q, X ₂ is a or L and X ₃ is Q, QSLVHX R, or F _{1 NX 2 X 3 TY (SEQ} ID NO: 180) the amino acid sequence of CDR-L1 sequence, comprising KVS (SEQ ID NO: 64) the CDR-L2 sequence of the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65) and the CDR-L3 sequence of the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and V _H3 and V _L3 form the third binding CD38 polypeptide Antigen binding site.

In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third polypeptide The chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; _VL2 is the variable domain of the second immunoglobulin light chain; _VL3 is the variable domain of the third immunoglobulin light chain; V _H1 is the first immunoglobulin Globulin heavy chain variable domain; V _H2 is the second immunoglobulin heavy chain variable domain; V _H3 is the third immunoglobulin heavy chain variable domain; _CL is the immunoglobulin light chain constant domain ; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 and C _H2 domains of an immunoglobulin hinge region; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said polypeptide of formula I and the formula II light chain polypeptides, form a staggered - Heavy chain pair; and wherein V _H1 and V _L1 form a first antigen binding site that binds to a CD28 polypeptide, wherein V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, wherein the V _H2 domain comprises: The CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) and those containing RGVYYALSPFDY (SEQ ID NO: 57) The CDR-H3 sequence of the amino acid sequence, and the _VL2 domain comprises: _{QSLVHX 1} NX ₂ X _{3 in which X 1} is E or Q, X ₂ is A or L, and X ₃ is Q, R or F The CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), the CDR-L2 sequence of the amino acid sequence of KVS (SEQ ID NO: 64), and the amine containing GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the base acid sequence, and where V _H3 and _VL3 form a third antigen binding site that binds to the CD38 polypeptide, for use in the amplification of virus-specific memory T cells.

In some embodiments, the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cells with the binding protein causes activation and/or proliferation of virus-specific memory T cells.

In some embodiments, provided herein is a method of expanding T cells, the method comprising contacting T cells in vitro or ex vivo with a binding protein, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites , Wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain includes a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III ] And the fourth polypeptide chain includes the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; 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 a third immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ Is an amino acid linker; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide, wherein V _H2 and V _L2 form a second antigen-binding site that binds to the CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and containing IKDKSNSYAT (SEQ ID NO: 55) ID NO: CDR-H3 sequence of 57 amino acid sequence), and the V _L2 domain comprises:: CDR-H2 sequence 56) and comprising the amino acid sequence RGVYYALSPFDY (SEQ ID NO containing wherein X ₁ is E or Q, X ₂ is A or L and X ₃ is Q, R or F. QSLVHX ₁ NX ₂ X ₃ CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), containing KVS (SEQ ID NO: 64) the CDR-L2 sequence of the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65) and the CDR-L3 sequence of the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein V _H3 and V _L3 form a third antigen that binds to the CD38 polypeptide Binding site.

In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third polypeptide The chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; _VL2 is the variable domain of the second immunoglobulin light chain; _VL3 is the variable domain of the third immunoglobulin light chain; V _H1 is the first immunoglobulin Globulin heavy chain variable domain; V _H2 is the second immunoglobulin heavy chain variable domain; V _H3 is the third immunoglobulin heavy chain variable domain; _CL is the immunoglobulin light chain constant domain ; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 and C _H2 domains of an immunoglobulin hinge region; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said polypeptide of formula I and the formula II light chain polypeptides, form a staggered - Heavy chain pair; and wherein V _H1 and V _L1 form a first antigen binding site that binds to a CD28 polypeptide, wherein V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, wherein the V _H2 domain comprises: The CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) and those containing RGVYYALSPFDY (SEQ ID NO: 57) The CDR-H3 sequence of the amino acid sequence, and the _VL2 domain comprises: _{QSLVHX 1} NX ₂ X _{3 in which X 1} is E or Q, X ₂ is A or L, and X ₃ is Q, R or F The CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), the CDR-L2 sequence of the amino acid sequence of KVS (SEQ ID NO: 64), and the amine containing GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the base acid sequence, and where V _H3 and _VL3 form a third antigen binding site that binds to the CD38 polypeptide, for use in a method for expanding T cells.

In some embodiments, the T cell is a memory T cell or an effector T cell. In some embodiments, the T cell displays a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding the CAR.

In some embodiments, provided herein is a method for treating chronic viral infections, the method comprising administering to an individual or patient in need an effective amount of a binding protein, wherein the binding protein comprises three antigen binding sites Where the first polypeptide chain comprises the structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain includes a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2- C _H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the second immunoglobulin _{V L3} is the variable domain of the third immunoglobulin light chain; V _H1 is the variable domain of the first immunoglobulin heavy chain; V _H2 is the second immunoglobulin heavy chain variable domains; V _H3 is a third immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 is an immunoglobulin protein C _H2 heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding to the CD28 polypeptide Site, where V _H2 and V _L2 form a second antigen binding site that binds to the CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), The CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) and the CDR-H3 sequence containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V _L2 domain contains: Where X ₁ is E or Q, X ₂ is A or L and X ₃ is Q, R or F, the CDR-L1 sequence of the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), containing KVS (SEQ ID NO: 64) the CDR-L2 sequence of the amino acid sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), and wherein V _H3 and V _L3 form the third antigen binding site that binds to the CD38 polypeptide point.

In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third polypeptide The chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; _VL2 is the variable domain of the second immunoglobulin light chain; _VL3 is the variable domain of the third immunoglobulin light chain; V _H1 is the first immunoglobulin Globulin heavy chain variable domain; V _H2 is the second immunoglobulin heavy chain variable domain; V _H3 is the third immunoglobulin heavy chain variable domain; _CL is the immunoglobulin light chain constant domain ; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 and C _H2 domains of an immunoglobulin hinge region; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said polypeptide of formula I and the formula II light chain polypeptides, form a staggered - Heavy chain pair; and wherein V _H1 and V _L1 form a first antigen binding site that binds to a CD28 polypeptide, wherein V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, wherein the V _H2 domain comprises: The CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) and those containing RGVYYALSPFDY (SEQ ID NO: 57) The CDR-H3 sequence of the amino acid sequence, and the _VL2 domain comprises: _{QSLVHX 1} NX ₂ X _{3 in which X 1} is E or Q, X ₂ is A or L, and X ₃ is Q, R or F The CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), the CDR-L2 sequence of the amino acid sequence of KVS (SEQ ID NO: 64), and the amine containing GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the base acid sequence, and where V _H3 and V _L3 form a third antigen binding site that binds to a CD38 polypeptide, the binding protein is used in a method of treating chronic viral infections, wherein the method includes Individuals or patients in need administer an effective amount The binding protein.

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

In some embodiments that can be combined with any of the other embodiments described herein, the memory T cell is a CD8+ or CD4+ memory T cell. In some embodiments, the memory T cells are central memory T cells (T _CM ) or effector memory T cells (T _EM ).

In some embodiments that can be combined with any of the other embodiments described herein, the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human milk Epidemic virus (HPV), Epstein-barr virus (EBV), human foam virus (HFV), herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2) .

In some embodiments that can be combined with any of the other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.

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

In some embodiments, provided herein is a kit that includes one, two, three, or four polypeptide chains of a binding protein according to any of the above embodiments. In some embodiments, the kit further includes instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, such as those described above.

In some embodiments, provided herein is a kit that includes one, two, three, or four polynucleotides according to any of the above embodiments. In some embodiments, provided herein is a set of polynucleotides comprising one, two, three, or four polynucleotides in the set of polynucleotides, the polynucleotides comprising: (a) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, the polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191 The third polynucleotide and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, comprising SEQ ID NO: 193 The second polynucleotide of the polynucleotide sequence of ID NO: 194, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 195, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 196 Polynucleotide; (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, comprising SEQ ID NO : The third polynucleotide of the polynucleotide sequence of 199 and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 200; (d) the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 201 A polynucleotide, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 203, and a third polynucleotide comprising SEQ ID NO: 204 The fourth polynucleotide of the polynucleotide sequence; (e) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 205, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 206 Nucleotides, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 207, and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 208; (f) comprising SEQ ID NO: The first polynucleotide of the polynucleotide sequence of 209, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 210, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 211 Acid and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 212; (g) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 213, the fourth polynucleotide comprising SEQ ID NO: 214 The second polynucleotide of the polynucleotide sequence, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 215, and the fourth polynucleotide including the polynucleotide sequence of SEQ ID NO: 216; (h) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 217, comprising SEQ ID NO The second polynucleotide of the polynucleotide sequence of: 218, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 219, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 220 (I) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 221, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 222, and including SEQ ID NO: 223 The third polynucleotide of the polynucleotide sequence of SEQ ID NO: 224 and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 224; (j) The first polynucleotide of the polynucleotide sequence of SEQ ID NO: 225 Nucleotide, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 227, and a polynucleotide comprising SEQ ID NO: 228 The fourth polynucleotide of the nucleotide sequence; (k) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 229, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 230 Acid, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 231, and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 232; (1) the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 233 The first polynucleotide of the polynucleotide sequence, the second polynucleotide including the polynucleotide sequence of SEQ ID NO: 234, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 235, and The fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 236; (m) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 237, the polynucleotide comprising SEQ ID NO: 238 The second polynucleotide of the acid sequence, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 239, and the fourth polynucleotide including the polynucleotide sequence of SEQ ID NO: 240; (n ) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 241, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 242, the polynucleotide sequence comprising SEQ ID NO: 243 And the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 244; (o) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 245, The second polynucleotide of the polynucleotide sequence of SEQ ID NO: 246, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 247, and the second polynucleotide of the polynucleotide sequence of SEQ ID NO: 248 Tetrapolynucleotide; (p) comprising SEQ ID NO: 249 The first polynucleotide of the nucleotide sequence, the second polynucleotide including the polynucleotide sequence of SEQ ID NO: 250, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 251, and The fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 252; (q) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 253, the polynucleotide comprising SEQ ID NO: 254 The second polynucleotide of the acid sequence, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 255, and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 256; (r ) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 257, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 258, the polynucleotide sequence comprising SEQ ID NO: 259 And the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 260; (s) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 261, The second polynucleotide of the polynucleotide sequence of SEQ ID NO: 262, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 263, and the second polynucleotide of the polynucleotide sequence of SEQ ID NO: 264 Four polynucleotide; (t) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 265, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 266, including SEQ ID The third polynucleotide of the polynucleotide sequence of NO: 267 and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 268; (u) the polynucleotide of the polynucleotide sequence of SEQ ID NO: 269 The first polynucleotide, the second polynucleotide including the polynucleotide sequence of SEQ ID NO: 270, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 271, and the third polynucleotide including SEQ ID NO: The fourth polynucleotide of the polynucleotide sequence of 272; or (v) the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 273, the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 274 Two polynucleotides, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 276.

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 become clear to those skilled in the art. These and other embodiments of the present invention are further described by the following specific embodiments.

Provided herein are trispecific and/or trivalent binding proteins, which comprise four polypeptide chains forming three antigen binding sites that specifically bind to one or more target proteins, wherein the The first pair of polypeptides of the binding protein possess dual variable domains with staggered orientation. General definition

Unless otherwise stated, as used herein, the following terms should be understood to have the following meanings. Unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular.

It should be understood that the aspects and embodiments of the disclosure described herein include "comprising" the aspect and the embodiment, "consisting of the aspect and the embodiment", and "essentially consisting of the aspect and the embodiment."

The term "polynucleotide" as used herein refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. In certain embodiments, the nucleotides constituting the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide. Such modifications include base modifications (such as bromouridine), ribose modifications (such as cytarabine and 2',3'-dodeoxyribose), and internucleotide linkage modifications (such as phosphorothioate, disulfide) Phosphorothioate, phosphoroselenoate, phosphorodiselenothioate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate). The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

An "isolated polynucleotide" refers to a polynucleotide of genome, cDNA, or synthetic origin or a certain combination thereof. The isolated polynucleotide: (1) does not differ from the polynucleotide in which it is found in nature. All or part of the acid is associated, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not exist as part of a larger sequence in nature.

An "isolated polypeptide" is a polypeptide that: (1) does not contain at least some other polypeptides normally found with it, (2) is substantially free of other polypeptides from the same source (for example, from the same species), (3) ) Expressed by cells from different species, (4) have been separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other substances associated with the isolated polypeptide in nature, (5) not related to Part of the polypeptide associated with the "isolated polypeptide" in nature is associated (through covalent or non-covalent interaction), (6) is operable with a polypeptide that is not associated with the isolated polypeptide in nature Ground association (through covalent or non-covalent interaction), or (7) does not exist in nature. This isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, has a synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other pollutants found in its natural environment that would interfere with its application (treatment, diagnosis, prevention, research or other applications).

Naturally occurring antibodies usually contain tetramers. Each such tetramer is usually composed of two identical polypeptide chain pairs, each pair having a full-length "light" chain (usually having a molecular weight of about 25 kDa) and a full-length "heavy" chain (usually having a molecular weight of about 50- 70 kDa molecular weight). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity for the target antigen. The amino terminal portion of each light chain and heavy chain generally includes a variable domain of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxy terminal part of each chain usually defines the constant domain responsible for effector functions. Thus, in the naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide comprising a variable domain (V _H) and three constant domains (C _H1, C _H2, and C _H3), wherein the V _H domain and the amino terminus of a polypeptide is located C _H3 domain at the carboxyl terminus, and full-length light chain immunoglobulin polypeptide comprising a variable domain (V _L) and a constant domain (C _L), wherein said V _L domain amine end is located polypeptide and the C _L domain is at the carboxy terminus.

Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has multiple sub-categories, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into multiple sub-categories, including but not limited to IgA1 and IgA2. Within the full-length light and heavy chains, the variable domain and constant domain are usually joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes about 10 or more amino groups The sour "D" zone. See, for example, Fundamental Immunology (Paul, W. editor, Raven Press, 2nd edition, 1989), which is incorporated by reference in its entirety for all purposes. The variable region of each light/heavy chain pair usually forms an antigen binding site. The variable domains of naturally occurring antibodies generally exhibit the same overall structure of relatively conserved framework regions (FR) joined by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs from the two chains of each pair are usually aligned by the framework regions, which can enable binding to specific epitopes. From the amino terminus to the carboxy terminus, both the light chain and heavy chain variable domains generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The term "CDR set" refers to a group of three CDRs that exist in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined in different ways according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda), Maryland (Md.) (1987) and (1991)) not only provides suitable antibodies The unambiguous residue numbering system for any variable region of the CDR also provides precise residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196 : 901-17; Chothia et al., 1989, Nature 342: 877-83) found that although there is significant diversity at the level of amino acid sequence, some sub-parts within Kabat CDR adopt almost the same peptide skeleton image These sub-parts are named L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate light chain and heavy chain regions, respectively. These regions can be called Chothia CDRs, which have the same characteristics as Kabat CDRs. Overlapping boundaries. The other boundaries of CDRs that overlap with Kabat CDRs have been defined 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. There are still other CDR boundary definitions that may not strictly follow one of the systems in this article, but will still overlap with Kabat CDR, but in view of specific residues or residue groups or even The entire CDR does not significantly affect the prediction of antigen binding or experimental findings, the other CDR boundaries may be shortened or lengthened. The method used herein can use the CDR defined according to any of these systems, but some examples use Kabat or Chothia Defined CDRs. The use of amino acid sequences to identify predicted CDRs is well known in the industry, as in the following literature: Martin, AC "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R. , Dübel S. Edited by Springer-Verlag, Berlin, pp. 33-51 (2010). Other conventional methods can also be used (for example, by combining with other known amino acid sequences in the variable regions of heavy and light chains). Compare to make sure Specify a region with sequence hyperdenaturation) to check the amino acid sequence of the heavy chain and/or light chain variable domain to identify the sequence of the CDR. The numbered sequences can be aligned visually or by using an alignment program (such as one of the CLUSTAL program suite), as described in the following literature: Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Conveniently use molecular models to correctly depict the framework and CDR regions, and thereby correct sequence-based alignments.

The term "Fc" as used herein refers to a molecule containing a sequence of a non-antigen-binding fragment derived from antibody digestion or produced by other means. The molecule is in the form of a monomer or a multimer, and the "Fc" may contain a hinge Area. Although the original immunoglobulin source of natural Fc is preferably of human origin and can be any immunoglobulin, IgG1 and IgG2 are preferred. Fc molecules are composed of monomeric polypeptides that can be linked into dimers or multimers by covalent (ie, disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomer subunits of natural Fc molecules ranges from 1 to 4, depending on the class (for example, IgG, IgA, and IgE) or subclass (for example, IgG1, IgG2, IgG3, IgA1 And IgGA2). An example of Fc is a disulfide-bonded dimer derived from papain digestion of IgG. The term "native Fc" as used herein is universal in monomer, dimer and multimer forms.

The F(ab) fragment usually includes a light chain and the V _H and _{CH1 domains of a heavy chain, wherein the V H-} C _H1 heavy chain part of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, F(ab) fragments can also include a light chain containing two variable domains separated by an amino acid linker, and two variable domains separated by an amino acid linker. And a heavy chain of a _{CH1 domain.}

F(ab') fragments usually include a light chain and a part of a heavy chain containing more constant regions (between the CH1 and CH2 domains), so that an interchain disulfide bond can be formed between the two heavy chains. The formation of F(ab')2 molecules.

As used herein, the term "binding protein" refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. Unless otherwise specified, the trispecific binding protein herein generally comprises four polypeptide chains forming at least three antigen binding sites, wherein the first polypeptide chain has a structure represented by the following formula: V _L2 -L ₁ -V _L1- L ₂ -C _L [I] and the second polypeptide chain has a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third The polypeptide chain has a structure represented by the following formula: V _H3 -C _H1 [III] and the fourth polypeptide chain has a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the first immunoglobulin Protein light chain variable domain; _VL2 is the second immunoglobulin light chain variable domain; _VL3 is the third immunoglobulin light chain variable domain; V _H1 is the first immunoglobulin heavy chain variable Domain; V _H2 is the variable domain of the second immunoglobulin heavy chain; V _H3 is the variable domain of the third immunoglobulin heavy chain; C _L is the constant domain of the immunoglobulin light chain; C _H1 is the immunoglobulin protein C _H1 heavy chain constant domain; and a hinge connects the C _H1 is an immunoglobulin hinge region and C _{H2 domain; L 1, L 2, L} 3 and L ₄ is a linker amino acid; and wherein The polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair.

A "recombinant" molecule is a molecule that has been prepared, expressed, produced, or isolated by recombinant means.

An example herein provides binding proteins that are biologically and immunologically specific for between one and three target antigens. Another embodiment herein provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment herein provides a performance vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Yet another embodiment herein provides a host cell that expresses such a binding protein (ie, comprises a nucleic acid molecule or vector that encodes a polypeptide chain that forms such a binding protein).

The term "exchangeability" as used herein refers to the interchangeability of variable domains within the binding protein form and while maintaining folding and final binding affinity. _{"Complete exchangeability" refers to the exchange of the V H1} and V _H2 domains in the polypeptide chain of Formula I or the polypeptide chain of Formula II while maintaining the complete functionality of the binding protein (as confirmed by the maintenance of binding affinity) The order of the two, and thus _{the ability to exchange the order of the VL1} and _VL2 domains (ie, reverse the order). Additionally, it should be noted that the name of V _H and V _L, only refers to the position on a particular domain of the protein chain in the form of the final form. For example, V _H1 and V _H2 can be derived from the V _L1 and V _L2 domains of the parent antibody and placed in the V _H1 and V _H2 positions of the binding protein. Similarly, V _L1 and V _L2 can be derived from the V _H1 and V _H2 domains in the parent antibody and placed in the V _H1 and V _H2 positions of the binding protein. Thus, V _H and V _L, the name refers to the position of the current location instead of the original parent antibodies. V _H and V _L domains therefore is "interchangeable."

As used herein, the term "antigen" or "target antigen" or "antigen target" refers to a molecule or part of a molecule that can be bound by a binding protein and can be used in animals to produce The antibody that binds to the epitope of the antigen. The target antigen may have one or more epitopes. Regarding each target antigen recognized by a binding protein, the binding protein can compete with a complete antibody that recognizes the target antigen.

The term "Her2" refers to human epidermal growth factor receptor 2, which is a member of the epidermal growth factor receptor family.

"CD3" is a differentiation factor cluster 3 polypeptide and a T cell surface protein, which is usually part of the T cell receptor (TCR) complex.

"CD28" is a cluster of differentiation 28 polypeptide and a T cell surface protein, which provides costimulatory signals for T cell activation and survival.

"CD38" is a cluster of differentiation 38 polypeptide, and is a glycoprotein found on the surface of many immune cells.

The term "T cell adaptor" is an indicator of the host's immune system (more specifically, the cytotoxic activity of T cells) and the binding protein for tumor target proteins.

The term "monospecific binding protein" refers to a binding protein that specifically binds to an antigen target.

The term "monovalent binding protein" refers to a binding protein with one antigen binding site.

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

The term "bivalent binding protein" refers to a binding protein with two binding sites.

The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

The term "trivalent binding protein" refers to a binding protein with three binding sites. In certain embodiments, the trivalent binding protein can bind to an antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

An "isolated" binding protein is a binding protein that has been identified and separated and/or recovered from a component of its natural environment. The pollutant components of its natural environment are substances that interfere with the diagnostic or therapeutic application of the binding protein, and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the binding protein is purified to: (1) greater than 95% by weight of antibody as determined by the Lowry method, and optimally greater than 99% by weight, (2) sufficient to pass The degree of the N-terminal or internal amino acid sequence of at least 15 residues measured by a rotating cup sequence analyzer, or (3) Coomassie blue or better by SDS-PAGE under reducing or non-reducing conditions It is silver stained and purified to homogeneity. The isolated binding protein includes the binding protein in situ within the recombinant cell because at least one component of the binding protein's natural environment will not be present.

As used herein, the term "substantially pure" or "substantially purified" refers to a compound that is the main species present (ie, in moles, which is more abundant than any other individual species in the composition) Or kind. In some embodiments, the substantially purified fraction is a composition in which the species account for at least about 50% (in moles) of all macromolecular species present. In other embodiments, a substantially pure composition will include greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In still other embodiments, the species is purified to be substantially uniform (the species of contaminants in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

The term "epitope" includes any determinant capable of specifically binding to immunoglobulin or T cell receptor, preferably a polypeptide determinant. In certain embodiments, epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphatidyl groups, or sulfonyl groups, and in certain embodiments, can have a specific three-dimensional Structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, when the binding protein preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules, it is said that the binding protein specifically binds to the antigen. In some embodiments, when the equilibrium dissociation constant ≤ 10 ^-8 M, more preferably when the equilibrium dissociation constant ≤ 10 ^-9 M, and optimally when the dissociation constant ≤ 10 ^-10 M, the binding protein specificity Binding antigen.

The dissociation constant (K _D ) of the binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis uses the BIAcore system (Pharmacia Biosensor; Piscataway, New Jersey (NJ)) to measure the ligand (target on the substrate of the biosensor) by using surface plasmon resonance (SPR) The instant binding interaction between antigen) and analyte (binding protein in solution). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on the matrix of the biosensor) and presenting the ligand (target antigen). As used herein, the term "K _D "refers to the dissociation constant of the interaction between a specific binding protein and the target antigen.

As used herein, the term "specific binding" refers to a binding protein or antigen-binding fragment thereof at least about 1 x 10 ^-6 M, 1 x 10 ^-7 M, 1 x 10 ^-8 M, 1 x 10 ^-9 M, The ability of 1 x 10 ^-10 M, 1 x 10 ^-11 M, 1 x 10 ^-12 M or greater Kd to bind to an epitope-containing antigen, and/or to compare the binding protein or antigen-binding fragment The affinity of a non-specific antigen is at least two times greater than the affinity to bind to an epitope.

In some embodiments, the antigen binding domains and/or binding proteins herein are associated with human and cynomolgus CD38 polypeptides (eg, CD38 extracellular domain, human CD38 subtype A, human CD38 subtype E, and cynomolgus CD38). Cross-reaction". When the EC50 for the two antigens is in a similar range, the binding protein that binds to antigen 1 (Ag1) "cross-reacts" with antigen 2 (Ag2). In this application, when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 20, the binding protein that binds to Ag1 cross-reacts with Ag2, and the affinity of the two antigens is measured by the same method.

As used herein, the term "linker" refers to one or more amino acids inserted between immunoglobulin domains to fold the light chain and heavy chain domains into staggered dual variable regions immunoglobulins to provide sufficient flexibility Residues. At the sequence level, linkers are inserted at the transition between variable domains or between variable domains and constant domains, respectively. Because the approximate size of immunoglobulin domains is well understood, transitions between domains can be identified. The exact position of the domain transition can be determined by locating peptide extensions that do not form secondary structure elements (such as β-folds or α-helices), as confirmed by experimental data or by techniques such as modeling or secondary structure prediction. assumed. The linker described herein is called L ₁ , which is located between the C-terminus _{of the V L2} domain on the light chain and the N-terminus of the _{V L1 domain; and L 2} , which is located at the C-terminus _{of the V L1 domain on the light chain} between the N-terminal and C _L domains. Re-coupling head is referred to as L _3, which is located between the N-terminus and C-terminus of domain V _H2 V _H1 domains; and N-terminal L _4, V _H2 domain located C-terminal domain and the C _Hl between.

As used herein, the term "vector" refers to any molecule (eg, nucleic acid, plastid, or virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plastid", which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thereby replicate with the host genome. In addition, certain vectors can direct the expression of genes that are operably linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "performance vectors"). Generally speaking, the expression vectors used in recombinant DNA technology are usually in the form of plastids. The terms "plastid" and "carrier" are used interchangeably in this article because plastid is the most commonly used form of carrier. However, this document is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cells or host cells are intended to refer not only to specific subject cells, but also to the progeny of such cells. Because certain modifications may occur in the offspring due to mutations or environmental influences, such offspring may actually be different from the parent cell, but such cells are still included in the scope of the term "host cell" as used herein. A wide 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). An example of a suitable bacterial expression vector is pUC19. In order to express the binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the binding protein, so that the polypeptide chain is expressed in the host cell and the polypeptide is preferably The chain is secreted into the medium in which the host cell is cultured, and the binding protein can be recovered from the medium.

The term "transformation" as used herein refers to a change in the genetic characteristics of a cell, and when a cell is modified to contain new DNA, the cell has been transformed. For example, if a cell is genetically modified from its natural state, the cell is transformed. After transformation, the transforming DNA can be physically integrated into the cell's chromosome to recombine with the cell's DNA, or can be maintained as an episomal element temporarily without being replicated, or can be replicated independently as a plastid. When DNA replicates as a cell divides, it is considered that the cell has been stably transformed. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and the cell has been "transfected" when the exogenous DNA has been introduced into the cell membrane. A variety of transfection techniques are well known in the industry. Such techniques can be used to introduce one or more foreign DNA molecules into suitable host cells.

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 existing in organisms (including viruses) that can be isolated from sources in nature and have not been intentionally modified by humans are naturally occurring. Similarly, "non-naturally occurring" as used herein refers to objects that are not found in nature or have been structurally modified or synthesized by humans.

As used herein, twenty conventional amino acids and their abbreviations follow conventional usage. Twenty kinds of stereoisomers of conventional amino acids (for example, D-amino acids); non-natural amino acids and analogs (such as α-, α-disubstituted amino acids, N-alkyl amino acids) , Lactic acid and other unconventional amino acids) can also be suitable components for binding protein polypeptide chains. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyl lysine, O -Phosphoserine, N-acetylserine, N-methionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and Other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide labeling method used herein, according to standard usage and conventions, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction.

Naturally occurring residues can be divided into multiple categories based on common side chain characteristics: (1) Hydrophobic: 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 hydrophobic: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues that affect chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

Conservative amino acid substitutions can involve the exchange of a member of one of these classes with another member of the same class. Non-conservative substitutions can involve the exchange of members of one of these classes with members of another class.

The skilled person will be able to use well-known techniques to determine suitable variants of the polypeptide chain of the binding protein. For example, those skilled in the art can identify suitable regions in the polypeptide chain that can be changed without destroying the activity by targeting regions that are not considered important for activity. Alternatively, those skilled in the art can identify residues and parts of the molecule that are conserved among similar polypeptides. In addition, even regions that may be important for biological activity or structure can undergo conservative amino acid substitutions without destroying biological activity or without adversely affecting the structure of the polypeptide.

As used herein, the term "patient" includes human and animal subjects.

The term "treatment" ("treatment" or "treat") as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those suffering from disorders as well as those susceptible to disorders or those to be prevented. In certain embodiments, the binding protein can be used to treat people with cancer or people who are susceptible to cancer, or to improve cancer in human subjects. The binding protein can also be used to prevent cancer in human patients. In certain embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, germinal center B cell lymphoma, or B cell Acute lymphoblastic leukemia. In other embodiments, the binding protein can be used to treat people with or susceptible to inflammatory disorders, or to improve inflammatory disorders in human subjects.

The term "pharmaceutical composition" or "therapeutic composition" as used herein refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

The terms "effective amount" and "therapeutically effective amount" when used with reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dose sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of the binding protein sufficient to inhibit one or more clinically defined pathological processes related to the condition being treated for a certain period of time. The effective amount can vary depending on the specific binding protein used, and also depends on various factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, those factors to be considered will include factors such as the patient's age, weight, and health status, as well as the dose-response curve and toxicity data obtained in preclinical animal work. The determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is well-versed by those skilled in the art.

One embodiment herein provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of binding protein. Trispecific and / or trivalent binding protein

Certain aspects herein relate to trispecific and/or trivalent binding proteins, which comprise four polypeptide chains forming three antigen binding sites that specifically bind to one or more target proteins, Wherein the first pair of polypeptides forming the binding protein possess dual variable domains with staggered orientation and where the second pair of polypeptides forming the binding protein possess a single variable domain. Any CDR or variable domain of any antigen binding protein described herein can be used in the trispecific binding protein herein.

In some embodiments, each of the three antigen binding sites binds a different target (eg, a polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains forming the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and The three polypeptide chains comprise the structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain comprises the structure represented by the following formula: V _L3 -C _L [IV] Where: _VL1 is the variable domain of the first immunoglobulin light chain; _VL2 is the variable domain of the second immunoglobulin light chain; _VL3 is the variable domain of the third immunoglobulin light chain; 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; _CL is the immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; hinge connection is C _H1 above hinge region and an immunoglobulin C _H2 domain; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said polypeptide of formula I and the formula II is formed interleaving polypeptide Light chain-heavy chain pair.

It is contemplated that any of the antigen binding sites described herein can be used in a trispecific binding protein herein, for example, comprising four polypeptide chains having the above-mentioned structure. For example, in some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site, and V H2} and V _L2 that form a second antigen binding site that binds a CD3 polypeptide. The domain pair and the V _H3 and _VL3 domain pair forming the third antigen binding site. In some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site that binds to a CD28 polypeptide, V H2} and V H2 that form a second antigen binding site that binds to a CD3 polypeptide The V _{L2 domain pair and the V H3} and V _L3 domain pair forming the third antigen binding site. In some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site, and a V H2} and V _L2 domain that forms a second antigen binding site that binds to a CD3 polypeptide _{Pair and form a V H3} and V _L3 domain pair that binds to the third antigen binding site of the tumor target protein. In some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site that binds to a CD28 polypeptide, V H2} and V H2 that form a second antigen binding site that binds to a CD3 polypeptide The V _{L2 domain pair and the V H3} and V _L3 domain pair forming the third antigen binding site that binds to the tumor target protein. In some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site that binds to a CD28 polypeptide, V H2} and V H2 that form a second antigen binding site that binds to a CD3 polypeptide The _{pair of V L2 domains and the pair of V H3} and V _L3 domains that form the third antigen binding site that binds to the CD38 polypeptide. In some embodiments, the trispecific binding protein herein comprises a _{pair of V H1} and V _{L1 domains that form a first antigen binding site that binds to a CD28 polypeptide, V H2} and V H2 that form a second antigen binding site that binds to a CD3 polypeptide The _{pair of V L2 domains and the pair of V H3} and V _L3 domains that form a third antigen binding site that binds to the HER2 polypeptide.

In some embodiments, the binding protein herein binds one or more tumor target proteins and one or more T cell target proteins. In some embodiments, the binding protein can specifically bind to two different epitopes on a tumor target protein and a single T cell target protein. In some embodiments, the binding protein can specifically bind to one tumor target protein and two different T cell target proteins (for example, 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 of the binding protein And the fourth polypeptide chain 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 can be used in any of the trispecific binding proteins described herein are provided in Table 1.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 156 The amino acid sequence is an amino acid sequence that is at least 95% identical; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth poly The peptide chain includes 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.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 160. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or the amino acid sequence of SEQ ID NO: 164. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 168. The amino acid sequence is an amino acid sequence that is at least 95% identical; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171.

In some embodiments, the binding protein herein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or the amino acid sequence of SEQ ID NO: 172. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 176 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 181 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain The peptide chain includes the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or the amino acid sequence of SEQ ID NO: 185. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.

In some embodiments, the binding protein herein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or the amino acid sequence of SEQ ID NO: 100. The amino acid sequence is at least 95% identical to an amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101 Base acid sequence; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 104 The amino acid sequence is an amino acid sequence that is at least 95% identical; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth poly The peptide chain includes 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.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 112. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113 Base acid sequence; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 116. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117 Base acid sequence; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or the amino acid sequence of SEQ ID NO: 120. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 124 An amino acid sequence that is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127.

In some embodiments, the binding protein herein comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or the amino acid sequence of SEQ ID NO: 128. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 132 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or the amino acid sequence of SEQ ID NO: 136. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or the amino acid sequence of SEQ ID NO: 140. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141 Base acid sequence; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 144 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145 The third polypeptide chain includes the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain The peptide chain includes the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 148 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149 Base acid sequence; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 152. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153 The third polypeptide chain includes the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain The peptide chain includes the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 286 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 287 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 287 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 288; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 289.

In some embodiments, the binding protein herein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 290 or the amino acid sequence of SEQ ID NO: 290. The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 291 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 291 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 292; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 293.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 294 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 295 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 295 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 296; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 297.

In some embodiments, the binding protein herein 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 the amino acid sequence of SEQ ID NO: 298 The amino acid sequence is at least 95% identical to the amino acid sequence; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 299 or an amine that is at least 95% identical to the amino acid sequence of SEQ ID NO: 299 Base acid sequence; the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 300; and the fourth poly The peptide chain includes the amino acid sequence of SEQ ID NO: 301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 301. Anti- CD38 binding site

Certain aspects herein relate to binding proteins that comprise 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 industry, and include, but are not limited to, the polypeptide represented by NCBI accession number NP_001766.2 or the polypeptide produced from NCBI gene ID number 952. In some embodiments, the antigen binding site binds to a human CD38 polypeptide, a non-human primate (eg, cynomolgus monkey) CD38 polypeptide, or a human CD38 polypeptide, and a non-human primate (eg, cynomolgus monkey) CD38 polypeptide. In some embodiments, the binding protein comprising the antigen binding site that binds to the CD38 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent or multispecific and/or Or multivalent.

In some embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding site described below can be used in monospecific antibodies.

In other embodiments, any of the CDRs and/or variable domains of the anti-CD38 binding site described below can be used for a trispecific binding protein comprising four polypeptides forming three antigen binding sites (for example, as described above). Mentioned) in any binding site. In certain embodiments, the binding protein comprising an antigen binding site that binds to a CD38 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites as described above, wherein the V _H3 and V _L3 domains Align and form a third antigen binding site that binds to the CD38 polypeptide.

Various characteristics of exemplary binding sites and binding proteins are described herein. For example, in some embodiments, the anti-CD38 binding site cross-reacts with human CD38 (eg, human CD38 subtype A and/or subtype E polypeptide) and cynomolgus CD38. In some embodiments, a binding protein comprising an anti-CD38 binding site induces apoptosis of CD38+ cells. In some embodiments, a binding protein comprising an anti-CD38 binding site recruits T cells to CD38+ cells and optionally activates T cells (eg, via TCR stimulation and/or costimulation).

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

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

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

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

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), and contains IWYDGSNK ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 38) and the CDR-H3 sequence of the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and/or the antibody light chain variable (VL) domain , Which includes the CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), the CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41) and the QQLNSFPYT (SEQ ID NO: 42) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), and contains IWYDGSNK ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 38) and the CDR-H3 sequence of the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and the antibody light chain variable (VL) domain, which Contains the CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), the CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41) and QGLNSFPYT (SEQ ID NO: 42) The CDR-L3 sequence of the amino acid sequence.

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

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least an amino acid sequence of at least 86% with QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGLRVTSASTAYMELSSLRSEDTAVYFCART NOV 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (at least 85% of SEQ ID NO: 86%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 79; and/or an amino acid containing SEQ ID NO: 80 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 79; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 80 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFD (SEQ ID NO: at least 85% SEQ ID: at least 85%, at least 85%) 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (at least 85%, at least 85% of SEQ ID NO: 82) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 81; and/or an amino acid containing SEQ ID NO: 82 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 81; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 82 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTL, at least 85% of SEQ ID: at least 85% of SEQ ID: 83VTV 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% of the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (at least 85% of SEQ ID NO: 86%, SEQ ID NO: 84) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 83; and/or an amino acid containing SEQ ID NO: 84 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 83; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 84 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDP IDQMNSLRAEDTAVYHCARDP IDQMNSLRAEDTAVYHCARDP IDQMNSLRAEDTAVYHCARDP IDG 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which comprises an amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (at least 85% of SEQ ID NO: 86%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 87; and/or an amino acid containing SEQ ID NO: 88 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 87; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 88 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLRAEDTAVYYCARMFRGVSS at least 85% SEQ ID NO: 89% SEQ ID NO: 89: 89% SEQ ID NO: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which comprises an amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (at least 85% of the amino acid sequence of SEQ ID NO: 86%, at least 85%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 89; and/or an amino acid containing SEQ ID NO: 90 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 89; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 90 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain, which comprises an amino acid sequence of at least 86% with QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSM, at least 85% SEQ ID: at least 85% WG acid sequence: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK at least 86% (SEQ ID NO: 86%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 85; and/or an amino acid containing SEQ ID NO: 86 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 85; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 86 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWG at least 85% of the amino acid sequence (SEQ ID NOKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWG, at least 85% SEQ ID: at least 277% SEQ ID: 86% TVSS) 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (at least 85% of SEQ ID NO: 278%, at least 85% of SEQ ID NO: 278%). %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 277; and/or an amino acid containing SEQ ID NO: 278 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 277; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 278 The antibody light chain variable (VL) domain. In some embodiments, the VH domain and/or VL domain are humanized.

In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGF VSSDY, at least 86% SEQ ID NO: VTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSM) at least 85% VT: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% of the amino acid sequence of DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPL 280% (SEQ ID NO: at least 86%, at least 280% SEQ IDLGVYLCSQSTHVPL280%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 279; and/or an amino acid containing SEQ ID NO: 280 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD38 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 279; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 280 The antibody light chain variable (VL) domain. In some embodiments, the VH domain and/or VL domain are humanized.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises 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 trispecific binding protein comprising four polypeptides containing three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain Chain pair (for example, as described herein). In some embodiments, any of the above anti-CD38 antigen-binding site of the VH and VL domains expressed V _H3 and V _L3 and the CD38 binding polypeptides form the third antigen binding site. In some embodiments, V _H1 and V _L1 form a first antigen binding site that binds to a CD28 polypeptide, and V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, as described above and/or Table 2. any anti-CD38 antigen-binding site of the VH and VL domains expressed V _H3 and V _L3 and the CD38 binding polypeptides form the third antigen binding site.

The sequences of exemplary anti-CD38 antigen binding sites are provided in Table 2. In some embodiments, the binding protein comprising the anti-CD38 antigen binding site herein comprises 1, 2, 3, 4, 5 or all 6 CDR sequences of the anti-CD38 antibodies described in Table 2. In some embodiments, the binding protein comprising the anti-CD38 antigen binding site herein comprises the VH domain sequence and/or VL domain sequence of the anti-CD38 antibody described in Table 2. Table 2. Anti-CD38 binding protein sequence. Sequence type molecular description SEQ ID NO sequence CDR Anti-CD38 (VH1) CDR-H1 13 GYTFTSYA CDR-H2 14 IYPGQGGT CDR-H3 15 ARTGGLRRAYFTY CDR-L1 16 QSVSSYGQGF CDR-L2 17 GAS CDR-L3 18 QQNKEDPWT Anti-CD38 (hhy992) CDR-H1 19 GYTLTEFS CDR-H2 20 FDPEDGET CDR-H3 twenty one TTGRFFDWF CDR-L1 twenty two QSVISRF CDR-L2 twenty three GAS CDR-L3 twenty four QQDSNLPIT Anti-CD38 (hyb5739) CDR-H1 25 GYAFTTYL CDR-H2 26 INPGSGST CDR-H3 27 ARYAYGY CDR-L1 28 QNVGTA CDR-L2 29 SAS CDR-L3 30 QQYSTYPFT Anti-CD38 (hyb6284) CDR-H1 31 GYSFTNYA CDR-H2 32 ISPYYGDT CDR-H3 33 ARRFEGFYYSMDY CDR-L1 34 QSLVHSNGNTY CDR-L2 35 KVS CDR-L3 36 SQSTHVPLT Anti-CD38 (hhy1195) CDR-H1 37 GFTFSSYG CDR-H2 38 IWYDGSNK CDR-H3 39 ARDPGLRYFDGGMDV CDR-L1 40 QGISSY CDR-L2 41 AAS CDR-L3 42 QQLNSFPYT Anti-CD38 (hhy1370) CDR-H1 43 GFTFSSYG CDR-H2 44 IWYDGSNK CDR-H3 45 ARMFRGAFDY CDR-L1 46 QGIRND CDR-L2 47 AAS CDR-L3 48 LQDYIYYPT Variable domain CD38 VH1 VH 79 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS VL 80 DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK CD38 hhy992 VH 81 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS VL 82 EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK CD38 hu5739 VH 83 QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS VL 84 DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK CD38 hu6284 VH 85 QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS VL 86 DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK CD38 hhy1195 VH 87 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS VL 88 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK CD38 hhy1370 VH 89 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS VL 90 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK CD38 hyb5739 VH 277 QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTLTVSS VL 278 DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK CD38 hyb6284 VH 279 QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSMDYWGQGTSVTVSS VL 280 DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK

This document further provides antibodies (eg, monospecific antibodies) comprising any of the anti-CD38 CDRs and/or variable domains described above.

In some embodiments, the binding protein herein comprises an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus CD38 polypeptide. Exemplary analyses for determining whether an antigen binding site binds an antigen are described herein and are known in the industry, including (but not limited to) ELISA, SPR, and flow cytometry analysis. Anti- HER2 binding site

Certain aspects herein relate to binding proteins that comprise an antigen binding site that binds to a HER2 polypeptide. 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 industry and include but are not limited to those 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 The polypeptide or the polypeptide produced from NCBI gene ID No. 2064. In some embodiments, the binding protein comprising 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 Or multivalent. In some embodiments, the binding protein comprising the antigen binding site that binds to the HER2 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites as described above, wherein the V _H3 and V _L3 domains are paired To form a third antigen binding site that binds to the HER2 polypeptide.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain, which comprises an amino acid sequence containing GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2) CDR-H1 sequence containing IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4) or IYPTNAYT (SEQ ID NO: 5) amino acid sequence and CDR-H2 sequence containing SRWGGDGFYAMDY (SEQ ID NO : 6), CDR-H3 sequence of the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7) or SRWGGSGFYAMDY (SEQ ID NO: 8); and/or antibody light chain variable (VL) domain, which contains QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10) amino acid sequence CDR-L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and QQHYTTP ( SEQ ID NO: 12) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain, which comprises an amino acid sequence containing GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2) CDR-H1 sequence containing IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4) or IYPTNAYT (SEQ ID NO: 5) amino acid sequence and CDR-H2 sequence containing SRWGGDGFYAMDY (SEQ ID NO : 6), CDR-H3 sequence of the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7) or SRWGGSGFYAMDY (SEQ ID NO: 8); and antibody light chain variable (VL) domain, which contains QDVNTA (SEQ ID NO: 8) ID NO: 9) or QDVQTA (SEQ ID NO: 10) amino acid sequence CDR-L1 sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11) and QQHYTTP (SEQ ID NO: 12) CDR-L3 sequence of the amino acid sequence.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the binding of HER2 binding site comprises: an antibody heavy chain variable (VH) domain that comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76) the amino acid sequence of 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 amino acid sequence; and/or antibody Light chain variable (VL) domain, which contains the same as DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQD The amino acid sequence of VQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78) 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an amino acid sequence containing SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76 An antibody heavy chain variable (VH) domain; and/or an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, the binding site that binds to HER2 comprises: an amino acid sequence containing SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76 An antibody heavy chain variable (VH) domain; and an antibody light chain variable (VL) domain containing the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGG: at least 85% of the acid sequence: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (at least 85% of SEQ ID NO: 77%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72; and/or an amino acid containing SEQ ID NO: 77 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 77 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGE: at least 85% of the amino acid sequence (SEQ ID NO: 85%, SEQ ID NO: 85%, at least 86% amino acid sequence: WGMNSLRAEDTAVYYCSRWGGE: VTYVSS) 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (at least 85% of SEQ ID NO: 77%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 73; and/or an amino acid containing SEQ ID NO: 77 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 73; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 77 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYVSSYWG at least 85% SEQ ID ID: at least 86% SEQ ID ID NO: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (at least 85% of SEQ ID NO: 77%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 75; and/or an amino acid containing SEQ ID NO: 77 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 75; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 77 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGS NOGGFYVSS: at least 85% of the acid sequence (SEQ ID NO: 85%), at least 85% of SEQ IDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGS NOV VSS: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (at least 85% of SEQ ID NO: 77%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 74; and/or an amino acid containing SEQ ID NO: 77 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 74; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 77 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYVSSYWG at least 86% (SEQ ID NO: 86%, at least 86% SEQ ID NO: 86%, at least 86% SEQ ID ID NO: 86%) and at least 86% 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (at least 85% of SEQ ID NO: 77%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 76; and/or an amino acid containing SEQ ID NO: 77 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 76; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 77 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGGTLQMNSLRAEDTAVYYCSRWGG: at least 85% (SEQ ID NO:85%), at least 85% of SEQID: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78%, at least 87%, at least 85% of the amino acid sequence) %, 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 amino acid sequence. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72; and/or an amino acid containing SEQ ID NO: 78 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 72; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 78 The antibody light chain variable (VL) domain.

In some embodiments, the anti-HER2 antigen binding site herein comprises 1, 2, 3, 4, 5, or all 6 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 herein comprises 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.

The sequences of exemplary anti-HER2 antigen binding sites are provided in Table 3. In some embodiments, the anti-HER2 antigen binding sites herein comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-HER2 antibodies described in Table 3. In some embodiments, the anti-HER2 antigen binding site herein comprises the VH domain sequence and/or VL domain sequence of the anti-HER2 antibody described in Table 3. Table 3. Anti-HER2 binding protein sequence. Sequence type molecular description SEQ ID NO sequence CDR Anti-Her2 (trastuzumab) heavy chain CDR CDR-H1 (original) 1 GFNIKDTY CDR-H1 30R 2 GFNIRDTY CDR-H2 (original) 3 IYPTNGYT CDR-H2 55Q 4 IYPTQGYT CDR-H2 56A 5 IYPTNAYT CDR-H3 (original) 6 SRWGGDGFYAMDY CDR-H3 102E 7 SRWGGEGFYAMDY CDR-H3 102S 8 SRWGGSGFYAMDY Anti-Her2 (trastuzumab) light chain CDR CDR-L1 (original) 9 QDVNTA CDR-L1 30Q 10 QDVQTA CDR-L2 (original) 11 SAS CDR-L3 (original) 12 QQHYTTP Variable domain Anti-Her2 trastuzumab and variant VH VH wt 72 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS VH 30R/55Q/102E 73 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS VH 30R/55Q/102S 74 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS VH 30R/56A/102S 75 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS VH 30R/56A/102E 76 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS Anti-Her2 trastuzumab and variant VL VL wt 77 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK VL 30Q 78 DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Other anti-tumor target binding sites

In some embodiments, the binding protein herein includes an antigen binding site that binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (for example, a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (for example, a human HER2 polypeptide). In some embodiments, tumor target proteins herein include, but are not limited to, 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 eosinophil chemokine), 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/eosinophil chemokine-2) , CCL25 (also known as TECK), CCL26 (also known as eosinophil chemokine-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, 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 (also known as ICOS), CD279 (also known as PD-1), 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, exonucleoside triphosphate diphosphate Phosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rβ, IL1, IL1A , IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL 9R, 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 Catenin), ITK, KIR, LAG3, LAMP1, Leptin, LPFS2, Class II MHC, MUC-1, NCR3LG1, NKG2D, NTPD-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152 , SISP1, SLC, SPG64, ST2 (also known as IL33 receptor), 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 of 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. Anti- CD28 binding site

Certain aspects herein relate to binding proteins that comprise 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 industry, and include, 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 from NCBI gene ID numbers 940 produced polypeptide. In some embodiments, the binding protein comprising the antigen binding site that binds to the CD28 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent or multispecific and/or Or multivalent. In some embodiments, the binding protein comprising the antigen binding site that binds to the CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of The antigen binding site binds to the CD3 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the CD38 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the HER2 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the tumor target protein.

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

In some embodiments, the binding site that binds to CD28 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCT IDWG at least 86% SEQ ID NOWG: at least 86% SEQ ID NOWG, at least 85% VTSISTAYMDVTTSS: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (at least 85% of SEQ ID acid sequence: 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 amino acid sequence. In some embodiments, the binding site that binds to CD28 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 91; and/or an amino acid containing SEQ ID NO: 92 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD28 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 91; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 92 The antibody light chain variable (VL) domain.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen binding site that binds to a tumor target protein (including 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 containing three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain Chain pair (for example, as described herein). In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent V _H1 and V _L1 and form the first antigen binding site that binds to the CD28 polypeptide. In some embodiments, the VH and VL domains of any anti-CD28 antigen binding site described above and/or in Table 4 represent V _H1 and V _L1 and form the first antigen binding site that binds to the CD28 polypeptide, V _H2 and _VL2 form a second antigen binding site that binds to the CD3 polypeptide, and V _H3 and _VL3 form a third antigen binding site that binds to tumor target proteins (including but not limited to CD38 or HER2).

The sequences of exemplary anti-CD28 antigen binding sites are provided in Table 4. In some embodiments, the anti-CD28 antigen binding sites herein comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-CD28 antibodies described in Table 4. In some embodiments, the anti-CD28 antigen binding site herein comprises the VH domain sequence and/or VL domain sequence of the anti-CD28 antibody described in Table 4. Table 4. Anti-CD28 binding protein sequence. Sequence type molecular description SEQ ID NO sequence CDR Anti-CD28 (sup) CDR-H1 49 GYTFTSYY CDR-H2 50 IYPGNVNT CDR-H3 51 TRSHYGLDWNFDV CDR-L1 52 QNIYVW CDR-L2 53 KAS CDR-L3 54 QQGQTYPY Variable domain Anti-CD28 (sup) VH 91 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS VL 92 DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK Anti- CD3 binding site

Certain aspects herein relate to binding proteins that comprise an antigen binding site that binds to a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, including CD3-δ (delta) (also known as T3D, IMD19, and CD3-δ (DELTA)), CD3-ε (also known as T3E, IMD18, and TCRE), and CD3-γ (gamma) (also known as T3G, IMD17 and CD3-γ (GAMMA)). Human CD3 polypeptides are known in the industry, and include, but are not limited to, polypeptides represented by NCBI accession numbers XP_006510029.1 or NP_031674.1 or polypeptides produced from NCBI gene ID numbers 915, 916, or 917. In some embodiments, the binding protein comprising the antigen binding site that binds to the CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent or multispecific and/or Or multivalent. In some embodiments, the binding protein comprising the antigen binding site that binds to the CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of The antigen binding site binds to the CD3 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the CD38 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the HER2 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, one of which binds to the CD28 polypeptide, and one of the antigens The binding site binds to the CD3 polypeptide, and one of the antigen binding sites binds to the tumor target protein.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or the antibody light chain variable (VL) domain , Which comprises a _{CDR- containing the amino acid sequence of QSLVHX 1} NX ₂ X ₃ TY (SEQ ID NO: 180) in _{which X 1} is E or Q, X ₂ is A or L and X ₃ is Q, R or F L1 sequence, CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the _{CDR-L1 sequence of the V L2} domain comprises 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 site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or the antibody light chain variable (VL) domain , Which includes the CDR-L1 sequence containing the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and the GQGTQYPFT (SEQ ID NO: 65) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and the antibody light chain variable (VL) domain, which Contains the CDR-L1 sequence containing the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the amino acid sequence.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or the antibody light chain variable (VL) domain , Which includes the CDR-L1 sequence containing the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and the GQGTQYPFT (SEQ ID NO: 65) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and the antibody light chain variable (VL) domain, which Contains the CDR-L1 sequence containing the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the amino acid sequence.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or the antibody light chain variable (VL) domain , Which includes the CDR-L1 sequence containing the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and the CDR-L2 sequence containing GQGTQYPFT (SEQ ID NO: 65) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and the antibody light chain variable (VL) domain, which Contains the CDR-L1 sequence containing the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the amino acid sequence.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and/or the antibody light chain variable (VL) domain , Which includes the CDR-L1 sequence containing the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and the CDR-L2 sequence containing GQGTQYPFT (SEQ ID NO: 65) CDR-L3 sequence of the amino acid sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and contains IKDKSNSYAT ( The CDR-H2 sequence of the amino acid sequence of SEQ ID NO: 56) and the CDR-H3 sequence of the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and the antibody light chain variable (VL) domain, which Contains the CDR-L1 sequence containing the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) and GQGTQYPFT (SEQ ID NO: 65) The CDR-L3 sequence of the amino acid sequence.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYALSPF, at least 85% of SEQ ID NO: at least 85% of the amino acid sequence (SEQ ID NO. 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 the amino acid sequence 100% identical; and / or an antibody light chain variable (VL) domain, which is selected from the group comprising DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98) at least 85%, at least 89%, at least 88%, at least 92%, at least 87% , 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 sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and/or comprises a variable domain selected from SEQ ID NO: 95, SEQ ID NO: 95, SEQ ID NO: 95, SEQ ID NO: 95, and SEQ ID NO: 95; The antibody light chain variable (VL) domain of the amino acid sequence of ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and containing a variable domain selected from SEQ ID NO: 95, SEQ ID NO : 96, SEQ ID NO: 97 and SEQ ID NO: 98 amino acid sequence of the antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYALSPF, at least 85% of SEQ ID NO: at least 85% of the amino acid sequence (SEQ ID NO. 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK, at least 85% of the amino acid sequence of SEQ ID NO: at least 86% (SEQ ID NO: 95%) %, 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 amino acid sequence. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and/or an amino acid containing SEQ ID NO: 95 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 95 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYALSPF, at least 85% of SEQ ID NO: at least 85% of the amino acid sequence (SEQ ID NO. 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK at least 86% (SEQ ID NO: 86%) %, 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 amino acid sequence. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and/or an amino acid containing SEQ ID NO: 96 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 96 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYALSPF, at least 85% of SEQ ID NO: at least 85% of the amino acid sequence (SEQ ID NO. 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% of the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGT: at least 87% (SEQ ID NO: at least 86%, at least 85% of the amino acid sequence of SEQ ID NO. %, 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 amino acid sequence. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and/or an amino acid containing SEQ ID NO: 97 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 97 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain, which comprises at least the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYALSPF, at least 85% of SEQ ID NO: at least 85% of the amino acid sequence (SEQ ID NO. 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% of the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK, at least 85% of the amino acid sequence of SEQ ID NO: at least 86% (SEQ ID NO: 98%) %, 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 amino acid sequence. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and/or an amino acid containing SEQ ID NO: 98 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 98 The antibody light chain variable (VL) domain.

In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising at least the amino acid sequence of at least 86% SEQ IDQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVGYY VSSDY, at least 86% SEQ IDQ NOVG VSSDY: 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 amino acid sequence; and/or antibody light chain variable (VL) domain, which contains at least 87% amino acid sequence with DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK, at least 85% of the amino acid sequence of SEQ ID NO: at least 86% (SEQ ID NO: 95%) %, 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 amino acid sequence. In some embodiments, the CD3 binding site comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 302; and/or an amino acid containing SEQ ID NO: 95 Sequence of the antibody light chain variable (VL) domain. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 302; and an antibody heavy chain variable (VH) domain containing the amino acid sequence of SEQ ID NO: 95 The antibody light chain variable (VL) domain.

In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen binding site that binds to a tumor target protein (including 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 containing three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain Chain pair (for example, as described herein). In some embodiments, any of the above antigen-binding site of the anti-CD3 VH and VL domains are V _H2 and V _L2 and forming a second antigen binding site binds to CD3 polypeptide. In some embodiments, V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide, and the VH and VL domains of any anti-CD3 antigen binding site described above and/or in Table 5 represent V _H2 and V _L2 and forms a second antigen binding site that binds to the CD3 polypeptide, and V _H3 and _VL3 form a third antigen binding site that binds to tumor target proteins (including but not limited to CD38 or HER2).

The sequences of exemplary anti-CD3 antigen binding sites are provided in Table 5. In some embodiments, the anti-CD3 antigen binding sites herein comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-CD3 antibodies described in Table 5. In some embodiments, the anti-CD3 antigen binding site herein comprises the VH domain sequence and/or VL domain sequence of the anti-CD3 antibody described in Table 5. Table 5. Anti-CD3 binding protein sequence. Sequence type molecular description SEQ ID NO sequence CDR Anti-CD3 (mid) CDR-H1 original 55 GFTFTKAW CDR-H2 original 56 IKDKSNSYAT CDR-H3 original 57 RGVYYALSPFDY CDR-L1 original 58 QSLVHNNANTY CDR-L1 QQ 59 QSLVHQNAQTY CDR-L1 ENLQ 60 QSLVHENLQTY CDR-L1 ENLF 61 QSLVHENLFTY CDR-L1 ENLR 62 QSLVHENLRTY CDR-L1 DNAQ 63 QSLVHDNAQTY CDR-L2 original 64 KVS CDR-L3 original 65 GQGTQYPFT CD3mid total CDR-L1 180 QSLVHX ₁ NX ₂ X ₃ TY, where X ₁ is E or Q, X ₂ is A or L and X ₃ is Q, R or F Variable domain Anti-CD3 (mid) VH 93 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS VL original 94 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VL 32/35 QQ 95 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VL ENLQ 96 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VL ENLF 97 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VL ENLR 98 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VL DNAQ 99 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHDNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK VH 185S 302 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS Linker

In some embodiments, the linkers L ₁ , L ₂ , L ₃ and L _{4 are} in the range of amino acid-free (length=0) to about 100 amino acid lengths, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linker can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. _{L 1} , L ₂ , L ₃ and L _{4 in} a binding protein may all have the same amino acid sequence or may all have different amino acid sequences.

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: ID NO: 71) and DKTHT (SEQ ID NO: 66), and those disclosed in International Publication Nos. WO2017/074878 and WO2017/180913. The examples listed above are not intended to limit the scope of this article in any way, and it has been shown that linkers containing randomly selected amino acids selected from the following are suitable in binding proteins: valine, leucine, iso Leucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, aspartic acid, glutamic acid, glycine and proline acid.

The identity and sequence of the amino acid residues in the linker can vary according to the type of secondary structure element that needs to be implemented in the linker. For example, glycine, serine, and alanine are best for linkers with the greatest flexibility. If a more rigid and extended linker is required, some combinations of glycine, proline, threonine and serine can be used. According to the required characteristics, any combination of amino acid residues and other amino acid residues can be regarded as linkers when necessary to construct larger peptide linkers.

In some embodiments, the length of L ₁ is at least twice _{the length of L 3.} In some embodiments, the length of L ₂ is at least twice _{the length of L 4.} In some embodiments, _{the length of L 1} is at least twice the length of L _{3 and} the length of L ₂ is at least twice the length of L ₄ . In some embodiments, L ₁ is 3 to 12 amino acid residues in length, L ₂ is 3 to 14 amino acid residues in length, and L ₃ is 1 to 8 amino acid residues in length. , And L ₄ is 1 to 3 amino acid residues in length. In some embodiments, L ₁ is 5 to 10 amino acid residues in length, L ₂ is 5 to 8 amino acid residues in length, and L ₃ is 1 to 5 amino acid residues in length. , And L ₄ is 1 to 2 amino acid residues in length. In some embodiments, L ₁ is the length of 7 amino acid residues, L ₂ is the length of 5 amino acid residues, L ₃ is the length of 1 amino acid residue, and L ₄ is 2. The length of the amino acid residues.

In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ are each independently the length of zero amino acids or comprise a sequence selected from: 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, L ₁ , L ₂ , L ₃ and L ₄ each independently comprise a sequence selected from: 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, L ₁ comprises the sequence GQPKAAP (SEQ ID NO: 67), L ₂ comprises the sequence TKGPS (SEQ ID NO: 68), L ₃ comprises the sequence S, and L ₄ comprises the sequence RT.

In some embodiments, _{at least one of L 1} , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO: 66). In some embodiments, L ₁ , L ₂ , L ₃ and L ₄ comprise the sequence DKTHT (SEQ ID NO: 66). Fc region and constant domain

In some embodiments, the binding protein described herein further comprises a second polypeptide chain comprising the Fc region is connected to the C _H1, the Fc region comprises an immunoglobulin hinge region and CH ₂ CH _3, and an immunoglobulin heavy chain constant area. In some embodiments, the binding protein described herein further comprises a third polypeptide chain comprising the Fc region is connected to the C _H1, the Fc region comprises an immunoglobulin hinge region and CH ₂ CH _3, and an immunoglobulin heavy chain constant area. In some embodiments, the binding protein described herein further comprises a second polypeptide chain comprising the Fc region is connected to the C _H1, the Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domain; and a third polypeptide chain comprising the Fc region further connected to the C _H1, said Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domain.

In some embodiments, the binding protein herein comprises a full-length antibody heavy chain or a polypeptide chain containing an Fc region. In some embodiments, the Fc region is a human Fc region, for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, Fc region comprises an antibody _{hinge, C H1, C H2, C} H3 , and optionally a C _H4 domain. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region contains one or more mutations described below. In some embodiments, the Fc region is the Fc region of one of the heavy chain polypeptides (eg, polypeptide 2 or 3) of the binding protein shown in Table 4. In some embodiments, the heavy chain constant region is the constant region of one of the heavy chain polypeptides (eg, polypeptide 2 or 3) of the binding protein shown in Table 4. In some embodiments, the light chain constant region is the constant region of one of the light chain polypeptides (eg, polypeptide 1 or 4) of the binding protein shown in Table 4.

In some embodiments, the binding protein herein includes one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from natural Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interaction with salvage receptors are known in the industry. Therefore, the term "Fc variant" may include molecules or sequences that are humanized from non-human natural Fc. In addition, the natural Fc contains a region that can be removed because it provides undesirable structural features or biological activity of the antibody-like binding protein of the present invention. Therefore, the term "Fc variant" encompasses molecules or sequences that lack one or more native Fc sites or residues, or where one or more Fc sites or residues have been modified, which sites or residues affect or Participate in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity after expression in selected host cells, (4) glycosylation, (5) ) Interaction with complement, (6) Binding to Fc receptors other than salvage receptors, or (7) Antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the binding protein (eg, trispecific binding protein) herein includes a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, the binding protein herein includes a "knob" mutation on the third polypeptide chain and a "hole" mutation on the second polypeptide chain. In some embodiments, the "knob" mutation comprises one or more substitutions 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 comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the "hole" mutation comprises one or more substitutions according to the EU index at positions corresponding to human IgG1 or IgG4 position 407 and optionally 349, 366, and/or 368. In some embodiments, the amino acid substitution is Y407V or Y407T and optionally Y349C, T366S, and/or L368A. In some embodiments, the "hole" mutations comprise substitutions at positions corresponding to 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.

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, wherein the The first Fc region contains one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4 position 366 and optionally 354 according to the EU index, wherein the amino acid substitution is T366W or T366Y and the The situation exists in S354C; and wherein the third polypeptide chain further comprises a second Fc region connected to CH1, and the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, Wherein the second Fc region contains one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4 position 407 and optionally 349, 366, and/or 368 according to the EU index, wherein the amine The base acid substitution is Y407V or Y407T and optionally Y349C, T366S and/or L368A.

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, wherein the The first Fc region contains one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4 position 407 and optionally 349, 366, and/or 368 according to the EU index, wherein the amino acid substitution Y407V or Y407T and optionally Y349C, T366S and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region connected to CH1, and the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises one or more amino acids at positions corresponding to human IgG1 or IgG4 at position 366 and optionally 354 according to the EU index Substitution, where the amino acid substitution is T366W or T366Y and optionally S354C.

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, wherein the The first Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a CH1 Connected second Fc region, the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises a human IgG1 or IgG4 according to the EU index. One or more amino acid substitutions at positions 366, 368, and/or 407 of, wherein the amino acid substitution is T366S, L368A, and/or Y407V.

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, wherein the The first Fc region contains one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4 positions 366, 368, and/or 407 according to the EU index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein said third polypeptide chain further comprises a second Fc region linked to CH1, said second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein said The second Fc region contains an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W.

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, wherein the The first Fc region contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain It further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises a region corresponding to an immunoglobulin hinge region according to the EU index. The amino acid substitutions at positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are 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 comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the The first Fc region contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and Wherein the third polypeptide chain further comprises a second Fc region connected to CH1, and the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region The region contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid The substitutions are S228P, S354C, T366W and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region, and the human IgG4 Fc region comprises Immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index The amino acid substitutions of, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, wherein The amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region The human IgG4 Fc region contains an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region contains positions 228, 354, 366 and corresponding to human IgG4 according to the EU index. The amino acid substitution at position 409, wherein the amino acid substitution is S228P, S354C, T366W, and R409K.

In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, wherein the amino acid The substitutions are F234A, L235A, S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region, and the human IgG4 Fc region comprises Immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region contains positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index The amino acid substitutions of, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, wherein The amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region The human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region includes positions 234, 235, 354 and 354 corresponding to human IgG4 according to the EU index. The amino acid substitution at position 366, wherein the amino acid substitution is F234A, L235A, S354C, and T366W.

In some embodiments, the binding protein herein contains one or more mutations to reduce effector functions, for example, Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/ Or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second Fc polypeptide chain further comprises a first region connected to the C _H1, the first Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domain; wherein said third polypeptide comprising a second Fc chain further connected to the C _H1 region, the second region comprises an immunoglobulin Fc hinge region and C _H2 and C _H3 immunoglobulin heavy chain constant domain; wherein The first and the second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each contain an amine at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index Amino acid substitutions, where the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise positions corresponding to positions 234 and 235 of human IgG1 according to the EU index. The amino acid substitutions at, where the amino acid substitutions are L234A and L235A. In some embodiments, the second Fc polypeptide chain further comprises a first region connected to the C _H1, the first Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domain; wherein said third polypeptide comprising a second Fc chain further connected to the C _H1 region, the second region comprises an immunoglobulin Fc hinge region and C _H2 and C _H3 immunoglobulin heavy chain constant domain; wherein The first and the second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each contain positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index The amino acid substitutions of, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise positions 234, 235, and 329 corresponding to human IgG1 according to the EU index. The amino acid substitution at the position of, wherein the amino acid substitution is L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise positions corresponding to positions 234 and 235 of human IgG4 according to the EU index The amino acid substitutions, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain, said second polypeptide chain Fc region further comprises a first connection to the C _Hl, the first Fc region comprises an immunoglobulin hinge region and C _H2 and C _H3 immunoglobulin heavy chain constant domain; and a third polypeptide chain, said chain further comprises a third polypeptide Fc region connected to the second C _H1, the second Fc region comprises an immunoglobulin hinge region and C _H2 and C _H3 immunoglobulin heavy chain constant domain; and wherein each of the first and second Fc region comprises a substitution at position 234 according to amino acid position 235 and corresponding to the EU index of human IgG4 , Wherein the amino acid substitutions are F234A and L235A.

In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, wherein The amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region The human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region includes positions 228, 234, 235, The amino acid substitutions at positions 349, 366, 368, 407, and 409, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region connected to CH1, wherein the first Fc region is a human IgG4 Fc region, and the human IgG4 Fc region includes an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domain, wherein the first Fc region comprises amino acids at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index Substitution, wherein the amino acid substitution is S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the first The second Fc region is a human IgG4 Fc region. The human IgG4 Fc region contains an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region contains a region corresponding to human IgG4 according to the EU index. The amino acid substitutions at positions 228, 234, 235, 354, 366, and 409 of, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.

In some embodiments, the Fc region is a human IgG4 Fc region, which contains one or more mutations that reduce or eliminate FcγI and/or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region, which contains one or more mutations that reduce or eliminate FcγI and/or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region, which contains amino acid substitutions 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 a human IgG4 Fc region, which contains amino acid substitutions 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 a human IgG4 Fc region, which contains 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 substitution is S228P, F234A, L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4 Fc region, which contains 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 the deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region, which contains amino acid mutations at the substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A and the deletion at 236; and/or R409K.

In some embodiments, the Fc region contains one or more mutations that reduce or eliminate Fc receptor binding and/or effector functions of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis ( ADCP), complement dependent cytotoxicity (CDC) and/or antibody dependent cytotoxicity (ADCC)).

In some embodiments, the Fc region is a human IgG1 Fc region, which contains 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 substitution is L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region, which contains 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 substitution is S298N, T299A, and/or Y300S.

In some embodiments, the binding protein herein contains one or more mutations to improve, for example, the stability of the hinge region and/or dimer interface of IgG4 (see, for example, Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutations comprise substitutions according to the EU index at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein further comprises a second polypeptide chain comprising the Fc region is connected to a first of the C _H1, the first Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant domain, and further comprising a second Fc C _H1 region connected to a third polypeptide chain, the second Fc region comprises an immunoglobulin hinge region and the C _H2 and C _H3 immunoglobulin heavy chain constant Domain; wherein said first and said second Fc regions are human IgG4 Fc regions; and wherein said first and said second Fc regions each comprise positions corresponding to positions 228 and 409 of human IgG4 according to the EU index The amino acid substitutions at, where the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein herein includes knob and hole mutations and one or more mutations for improving stability. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the Fc region is a human IgG1 Fc region, which contains 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 substitution is L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region, which contains 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 substitution is S298N, T299A, and/or Y300S. Nucleic Acid

Other aspects herein relate to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.

Other aspects herein relate to sets of polynucleotides, such as one or more polypeptides encoding binding proteins as described herein. In some embodiments, the set of polynucleotides herein includes one, two, three, or four polynucleotides in the set of polynucleotides, the polynucleotides comprising: (a) comprising SEQ ID The first polynucleotide of the polynucleotide sequence of NO: 189, the second polynucleotide of the polynucleotide sequence of SEQ ID NO: 190, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 191 Nucleotides and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, comprising SEQ ID NO: The second polynucleotide of the polynucleotide sequence of 194, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 195, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 196 Acid; (c) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, the polynucleotide comprising SEQ ID NO: 199 The third polynucleotide of the polynucleotide sequence and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 200; (d) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 201 Nucleotide, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 202, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 203, and the polynucleotide comprising SEQ ID NO: 204 The fourth polynucleotide of the acid sequence; (e) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 205, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 206 , The third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 207 and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 208; (f) the polynucleotide comprising SEQ ID NO: 209 The first polynucleotide of the nucleotide sequence, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 210, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 211, and the third polynucleotide comprising The fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 212; (g) the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 213, the polynucleotide of SEQ ID NO: 214 The second polynucleotide of the sequence, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 215, and the fourth polynucleotide including the polynucleotide sequence of SEQ ID NO: 216; (h) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 217, comprising SEQ ID NO: 21 The second polynucleotide of the polynucleotide sequence of 8, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 219, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 220 Acid; (i) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 221, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 222, the polynucleotide comprising SEQ ID NO: 223 The third polynucleotide of the polynucleotide sequence and the fourth polynucleotide including the polynucleotide sequence of SEQ ID NO: 224; (j) the first polynucleotide including the polynucleotide sequence of SEQ ID NO: 225 Nucleotide, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 226, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 227, and the polynucleotide comprising SEQ ID NO: 228 The fourth polynucleotide of the acid sequence; (k) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 229, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 230 , The third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 231 and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 232; (1) the polynucleotide comprising SEQ ID NO: 233 The first polynucleotide of the nucleotide sequence, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 234, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 235, and the third polynucleotide comprising The fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 236; (m) the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 237, the polynucleotide of SEQ ID NO: 238 The second polynucleotide of the sequence, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 239, and the fourth polynucleotide including the polynucleotide sequence of SEQ ID NO: 240; (n) The first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 241, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 242, the polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 243 The third polynucleotide and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 244; (o) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 245, comprising SEQ ID NO: 245 The second polynucleotide of the polynucleotide sequence of ID NO: 246, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 247, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 248 Polynucleotide; (p) comprises the polynucleotide sequence of SEQ ID NO: 249 The first polynucleotide of the column, the second polynucleotide including the polynucleotide sequence of SEQ ID NO: 250, the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 251, and the third polynucleotide including the polynucleotide sequence of SEQ ID NO: 251 The fourth polynucleotide of the polynucleotide sequence of NO: 252; (q) the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 253, the polynucleotide of the polynucleotide sequence of SEQ ID NO: 254 The second polynucleotide, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 255, and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 256; (r) comprising SEQ ID NO: 256 The first polynucleotide of the polynucleotide sequence of ID NO: 257, the second polynucleotide of the polynucleotide sequence of SEQ ID NO: 258, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 259 Polynucleotide and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 260; (s) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 261, comprising SEQ ID NO The second polynucleotide of the polynucleotide sequence of 262, the third polynucleotide of the polynucleotide sequence of SEQ ID NO: 263, and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 264 Glycolic acid; (t) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 265, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 266, comprising SEQ ID NO: 267 The third polynucleotide of the polynucleotide sequence of SEQ ID NO: 268 and the fourth polynucleotide of the polynucleotide sequence of SEQ ID NO: 268; (u) the first polynucleotide of the polynucleotide sequence of SEQ ID NO: 269 Nucleotide, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 270, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 271, and the polynucleotide comprising SEQ ID NO: 272 The fourth polynucleotide of the nucleotide sequence; or (v) the first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 273, the second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 274 Glycolic acid, the third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 275, and the fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 276.

Other aspects herein relate to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any binding protein described herein. 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, and a third vector encoding a third polypeptide chain of the binding protein And the fourth vector encoding the fourth polypeptide chain of the binding protein, as shown in the polynucleotides in Table 6. In some embodiments, the vector system includes a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system includes a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system includes a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the 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. The one or more vectors of the vector system can be any of the vectors described herein. In some embodiments, the 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, such as one, two, three, or four expression vectors.

Standard recombinant DNA methods 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 edition). The enzyme reaction and purification techniques can be carried out according to the manufacturer's instructions, as commonly achieved in the industry or as described herein. Unless specific definitions are provided, the terms used in analytical chemistry, synthetic organic chemistry, and medicine and medicinal chemistry described herein and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are well known in the industry. And the commonly used ones. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and treatment of patients.

In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of the nucleic acid sequence encoding the binding protein. Promoter may refer to a nucleic acid control sequence that directs the transcription of nucleic acid. When the first nucleic acid sequence is placed in a position having a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence are operably linked. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to the coding sequence of the binding protein. Examples of promoters may include, but are not limited to, from viruses (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, B Hepatitis B virus, simian virus 40 (SV40, etc.) genome-derived promoters, heterologous eukaryotic promoters (such as actin promoter, immunoglobulin promoter, heat shock promoter, etc.), CAG promoter ( Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK) promoter, tetracycline inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005) , Lac system, trp system, tac system, trc system, the main operon and promoter region of bacteriophage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase and the promoter of yeast α mating factor. The polynucleotide encoding the binding protein herein may be under the control of the following promoters: a constitutive promoter, an inducible promoter, or any other suitable promoters described herein, or those familiar with the art will easily recognize Other suitable promoters.

In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is a performance vector. The expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other performance control elements (eg, polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences derived from mammalian genes (such as globulin, elastase, albumin, α-fetoprotein, insulin, etc.), and enhancer sequences derived from eukaryotic viruses ( Such as the SV40 enhancer downstream of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer downstream of the replication origin, adenovirus enhancer, etc.). Examples of suitable vectors may include, for example, plastids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis-viral, measles, herpes virus, lentivirus, Retroviruses, 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. Biologically functional viruses and plastid DNA vectors that can be expressed and replicated in the host are known in the industry and can be used to transfect any cell of interest. Host cell

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

Other aspects herein relate to methods of producing any of the binding proteins described herein. In some embodiments, the method includes: a) culturing an isolated nucleic acid, vector, and/or vector system (for example, any isolated nucleic acid, vector, and/or described herein) under conditions such that the host cell expresses the binding protein Or vector system) host cell (for example, any host cell described herein); and b) isolating the binding protein from the host cell. The method of culturing host cells under conditions that express proteins is well known to those skilled in the art. Methods for separating proteins from cultured host cells are 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). ). Pharmaceutical composition

A therapeutic or pharmaceutical composition comprising a binding protein is within the scope of this document. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of binding protein or binding protein-drug conjugate mixed with a selected pharmaceutically or physiologically acceptable formulation suitable for the mode of administration.

Acceptable formulation materials are non-toxic to recipients at the dose and concentration used.

The pharmaceutical composition may contain for improving, maintaining or maintaining, for example, the pH, molar osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or Permeable blending materials. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamic acid, aspartic acid, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or lysine). Sodium bisulfate), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylene two Aminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides and other carbohydrates (Such as glucose, mannose or dextrin), protein (such as serum albumin, gelatin or immunoglobulin), coloring agents, flavoring and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low Molecular weight peptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine , Sorbic acid or hydrogen peroxide), solvents (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronic (Pluronics); PEG; sorbitan ester; polysorbate (such as polysorbate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol or tyloxapol ( tyloxapal), stability enhancers (such as sucrose or sorbitol), tonicity modifiers (such as alkali metal halides (such as sodium chloride or potassium chloride) or mannitol sorbitol), delivery vehicles, diluents, excipients Excipients and/or medical adjuvants (see, for example, Remington's Pharmaceutical Sciences (18th edition, AR Gennaro editor, Mack Publishing Company 1990) and subsequent versions, which are incorporated herein by reference for any purpose).

The optimal pharmaceutical composition will be determined by the skilled person according to, for example, the planned route of administration, delivery form, and required dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

The main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, the vehicle or carrier suitable for injection may be water, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0 to 8.5 or acetate buffer at about pH 4.0 to 5.5, which may further include sorbitol or a suitable substitute. In one embodiment herein, the binding protein composition can be prepared for storage by mixing the selected composition with the desired purity and optional formulations in the form of a lyophilized cake or an aqueous solution. In addition, the binding protein can be formulated as a lyophilized product using an appropriate excipient such as sucrose.

The pharmaceutical composition herein can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the composition may be selected for inhalation or for delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the technical scope of the industry.

The components of the formulation are present in concentrations that are acceptable at the site of administration. For example, a buffer is used to maintain the composition at a physiological pH or a slightly lower pH, usually in the pH range of about 5 to about 8.

When considering parenteral administration, the therapeutic composition used may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile isotonic solution that is properly preserved. Yet another preparation may involve the use of agents that provide controlled or sustained release of the product (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes). Molecules, which can then be delivered via depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting a long-lasting duration in the loop. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

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

It is also contemplated that certain formulations can be administered orally. In an embodiment herein, the binding protein administered in this manner can be formulated with or without those carriers commonly used in the compounding of solid dosage forms (such as tablets and capsules). For example, the capsule can be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional reagents may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used.

Another pharmaceutical composition may contain an effective amount of binding protein mixed with non-toxic excipients suitable for the manufacture of lozenges. Solutions can be prepared in unit dose form by dissolving the lozenge in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate , Stearic acid or talc.

The additional pharmaceutical compositions herein will be obvious to those skilled in the art, which include formulations that include binding proteins in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles (eg films) or microcapsules. The sustained release matrix may include polyester, hydrogel, polylactide, copolymer of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), Ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the industry.

The pharmaceutical composition to be used for in vivo administration must generally be sterile. This can be achieved by filtration through a sterile filter membrane. In the case of freeze-drying the composition, this method can be used for sterilization before or after freeze-drying and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in solution form. In addition, the parenteral composition is usually placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (eg, lyophilized).

This document also encompasses kits for producing a single dosage administration unit. The kit may each contain both a first container with dry protein and a second container with an aqueous formulation. Also included within the scope of this document are sets containing single-chamber and multi-chamber pre-filled syringes (for example, liquid syringes and lyosyringe).

The effective amount of the binding protein pharmaceutical composition used in treatment will depend on, for example, the treatment context and goals. Those familiar with the art will understand that the appropriate dosage level for treatment will therefore vary in part according to the following: the molecule delivered, the indication for which the binding protein is used, the route of administration, and the size of the patient (body weight, body surface, or organ size) And condition (age and general health). Therefore, clinicians can titrate the dose and change the route of administration to obtain the best therapeutic effect.

The frequency of dosing will depend on the pharmacokinetic parameters of the binding protein in the formulation used. Generally, the clinician will administer the composition until the dose to achieve the desired effect is reached. Therefore, the composition can be administered as a single dose, as two or more doses over time (they may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. The further improvement of the appropriate dosage is carried out in a conventional manner by those skilled in the art, and within the scope of tasks routinely performed by those skilled in the art. The appropriate dose can be determined by using appropriate dose response data.

The route of administration of the pharmaceutical composition is consistent with known methods, for example, oral; via intravenous, intraperitoneal, intracerebral (intracerebral), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Route injection; through a sustained release system; or through an implanted device. Where necessary, the composition can be administered by bolus injection, or continuously by infusion, or by implanting a device.

The composition can also be administered locally through the implantation of a membrane, sponge or other suitable material on which the desired molecule has been adsorbed or encapsulated. In the case of an implanted device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out via diffusion, timed release bolus injection, or continuous administration.

The pharmaceutical composition can be used to prevent and/or treat HIV infection. The pharmaceutical composition can be used as a standalone therapy or in combination with standard antiretroviral therapy.

This document also relates to kits that include binding proteins and other reagents that can be used to detect target antigen levels in biological samples. Such reagents can include detectable labels, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit includes a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be made of various materials such as glass or plastic. The container contains a composition that is effective in treating, preventing, and/or diagnosing conditions (for example, HIV infection) by itself or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous infusion bag or have a subcutaneous injection Vial with a stopper pierced by a needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and/or treat the selected condition. Alternatively or additionally, the article or kit may further include a second (or third) container, the second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. It can further include other materials required from a commercial and user perspective, including other buffers, diluents, screening programs, needles and syringes. Methods and uses of binding proteins Viruses

Certain aspects herein relate to methods for the expansion of virus-specific memory T cells. In some embodiments, the method includes contacting a virus-specific memory T cell with a binding protein herein, the binding protein is, for example, comprising a first antigen binding site that binds to a CD28 polypeptide, a second antigen binding site that binds to a CD3 polypeptide Site and a trispecific binding protein that binds to the third antigen binding site of the CD38 polypeptide.

In some embodiments, the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo.

In some embodiments, contacting the virus-specific memory T cells with the binding protein causes activation and/or proliferation of virus-specific memory T cells.

Other aspects herein relate to methods of expanding T cells. In some embodiments, the method includes contacting T cells with a binding protein herein, the binding protein, for example, comprising a first antigen binding site that binds to a CD28 polypeptide, a second antigen binding site that binds to a CD3 polypeptide, and a binding protein. A trispecific binding protein of the third antigen binding site of the CD38 polypeptide.

In some embodiments, the T cell is a memory T cell or an effector T cell.

In some embodiments, the T cell displays a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding the CAR.

Other aspects herein relate to, for example, methods of treating chronic viral infections in individuals in need. In some embodiments, the method includes administering to an individual in need an effective amount of the binding protein herein, the binding protein is, for example, comprising a first antigen binding site that binds to a CD28 polypeptide, and a second antigen that binds to a CD3 polypeptide. A binding site and a trispecific binding protein that binds to the third antigen binding site of the CD38 polypeptide.

In some embodiments, the individual is a human.

In some embodiments, the binding protein is administered to the individual in the form of a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.

In some embodiments, the administration of the binding protein results in the activation and/or proliferation of virus-specific memory T cells in the individual.

In any of the above methods, the memory T cells can be CD8+ or CD4+ memory T cells. In any of the above methods, the memory T cells can be central memory T cells (T _CM ) or effector memory T cells (T _EM ). cancer

Certain aspects herein relate to methods for preventing and/or treating cancer in a patient. In some embodiments, the method includes administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition herein.

In some embodiments, the binding protein herein is administered to patients in need for the treatment or prevention of cancer. In some embodiments, this document relates to methods of preventing and/or treating proliferative diseases or disorders (eg, cancer). In some embodiments, the method includes administering to the patient a therapeutically effective amount of at least one of the binding protein described herein or a pharmaceutical composition related thereto. In some embodiments, this document relates to the use of at least one of the binding proteins described herein or pharmaceutical compositions related thereto for the prevention and/or treatment of proliferative diseases or disorders (such as cancer) in patients in need thereof. In some embodiments, this document relates to at least one of the binding protein described herein or a pharmaceutical composition related thereto, for use in the manufacture for the prevention and/or treatment of proliferative diseases or disorders in patients in need thereof (e.g., , Cancer) used in drugs. In some embodiments, the patient is a human.

In some embodiments, the at least one binding protein is administered in combination with (or to be combined with) one or more anti-cancer therapies (for example, any anti-cancer therapies known in the industry, such as chemotherapeutic agents or therapies). In some embodiments, the at least one binding protein is administered before (or to be administered before) one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered simultaneously with (or to be administered with) one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered after (or to be administered after) one or more anti-cancer therapies.

In some embodiments, the binding protein comprises one or two antigen binding sites that bind to a T cell surface protein and another antigen binding site that binds to the extracellular domain of a human HER2 polypeptide. In some embodiments, the binding protein comprises an antigen binding site that binds to the extracellular domain of a human HER2 polypeptide, an antigen binding site that binds to a human CD28 polypeptide, and an antigen binding site that binds to a human CD3 polypeptide.

In some embodiments, cancer cells from the individual express HER2. In some embodiments, patients are selected for treatment based on cancer-based cells expressing human HER2 polypeptides. The analysis known in the industry to be suitable for detecting the expression of HER2 of cancer cells includes, but is not limited to, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis.

In some embodiments, the cancer (eg, HER2-positive cancer) is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

In some embodiments, the binding protein comprises one or two antigen binding sites that bind to a T cell surface protein and another antigen binding site that binds to the extracellular domain of a human CD38 polypeptide. In some embodiments, the binding protein comprises an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide, an antigen binding site that binds to a human CD28 polypeptide, and an antigen binding site that binds to a human CD3 polypeptide.

In some embodiments, cancer cells from the individual express CD38. In some embodiments, cancer cells express human CD38 subtype A polypeptides on their cell surface. In some embodiments, cancer cells express human CD38 subtype E polypeptides on their cell surface. In some embodiments, cancer-based cells express human CD38 subtype E polypeptides on their cell surfaces to select patients for treatment. In some embodiments, cancer cells express CD38 and CD28. In some embodiments, cancer cells express CD38 but not CD28.

In some embodiments, the cancer (eg, CD38 positive cancer) is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer , Germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

In certain embodiments, the cancer is multiple myeloma. Anti-CD38 antibodies have been tested for the treatment of multiple myeloma such as darlimumab. However, although multiple myeloma is considered treatable, almost all patients inevitably relapse, leading to the development of refractory diseases. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with previous multiple myeloma treatment. In some embodiments, the binding protein herein is administered to the patient as a first-line, second-line, or third-line treatment for multiple myeloma. Without wishing to be bound by theory, it is believed that the anti-CD38x anti-CD28x anti-CD3 binding protein herein can be used to treat multiple myeloma, for example, by recruiting T cells to tumor cells via anti-CD38 (or anti-CD28/anti-CD38), T cells engaged via anti-CD3/anti-CD28 activation and/or kill tumor cells through a perforin/granzyme-based mechanism. It has been reported that CD28 is a new cancer marker for multiple myeloma. See Nair, J.R. et al. (2011) J. Immunol. 187:1243-1253.

Any binding protein described herein can be used in the methods herein.

In some embodiments of any of the methods herein, the patient has been treated with darlimumab before administering the binding protein. As described herein, provided herein are anti-CD38 binding proteins and sites that do not compete with darlimumab for binding to CD38. Without wishing to be bound by theory, it is believed that this is advantageous, because patients previously treated with darlimumab can be treated with the binding protein herein, for example, without a washout period prior to treatment.

The binding protein can be used in any known analytical methods for detecting and quantifying one or more target antigens, such as competitive binding analysis, direct and indirect sandwich analysis, and immunoprecipitation analysis. The binding protein will bind the one or more target antigens with an affinity suitable for the analytical method used.

In certain embodiments, for diagnostic applications, the binding protein can be labeled with a detectable moiety. The detectable part may be any part capable of directly or indirectly generating a detectable signal. For example, the detectable moiety can be a radioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I, ⁹⁹ Tc, ¹¹¹ In, or ⁶⁷ Ga; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate , Rhodamine or luciferin; or enzymes such as alkaline phosphatase, β-galactosidase or horseradish peroxidase.

The binding protein can also be used for in vivo imaging. The binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host can be determined. The binding protein can be labeled with any part that can be detected by nuclear magnetic resonance, radiology, or other detection methods known in the industry in animals.

In certain embodiments, for clinical or research applications, the binding protein can be conjugated with a cytotoxic agent. Various antibodies conjugated to cytotoxic agents (ie, antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate the agent to the antibody are known in the industry; see, for example, Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel .Ther.11:2265-2276. Binding protein therapeutic composition and its administration

A therapeutic or pharmaceutical composition comprising a binding protein is within the scope of this document. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of binding protein or binding protein-drug conjugate mixed with a selected pharmaceutically or physiologically acceptable formulation suitable for the mode of administration. These pharmaceutical compositions can be used in any of the methods and uses described herein (eg, ex vivo, in vitro, and/or in vivo).

Acceptable formulation materials are preferably non-toxic to recipients at the dosage and concentration used.

The pharmaceutical composition may contain for improving, maintaining or maintaining, for example, the pH, molar osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or Permeable blending materials. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamic acid, aspartic acid, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or lysine). Sodium bisulfate), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylene two Aminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides and other carbohydrates (Such as glucose, mannose or dextrin), protein (such as serum albumin, gelatin or immunoglobulin), coloring agents, flavoring and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low Molecular weight peptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine , Sorbic acid or hydrogen peroxide), solvents (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronic (Pluronics); PEG; sorbitan ester; polysorbate (such as polysorbate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol or tyloxapol), stability Enhancers (such as sucrose or sorbitol), tonicity modifiers (such as alkali metal halides (such as sodium chloride or potassium chloride) or mannitol sorbitol), delivery vehicles, diluents, excipients and/or drugs Adjuvants (see, for example, Remington's Pharmaceutical Sciences (18th edition, AR Gennaro editor, Mack Publishing Company 1990) and subsequent versions, which are incorporated herein by reference for any purpose).

The optimal pharmaceutical composition will be determined by the skilled person according to, for example, the planned route of administration, delivery form, and required dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

The main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, the vehicle or carrier suitable for injection may be water, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0 to 8.5 or acetate buffer at about pH 4.0 to 5.5, which may further include sorbitol or a suitable substitute. In one embodiment herein, the binding protein composition can be prepared for storage by mixing the selected composition with the desired purity and optional formulations in the form of a lyophilized cake or an aqueous solution. In addition, the binding protein can be formulated as a lyophilized product using an appropriate excipient such as sucrose.

The pharmaceutical composition herein can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the composition may be selected for inhalation or for delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the technical scope of the industry.

The formulated components are present in a concentration acceptable to the site of administration. For example, a buffer is used to maintain the composition at a physiological pH or a slightly lower pH, usually in the pH range of about 5 to about 8.

When considering parenteral administration, the therapeutic composition used may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile isotonic solution that is properly preserved. Yet another preparation may involve the use of agents that provide controlled or sustained release of the product (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes). Molecules, which can then be delivered via depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting a long-lasting duration in the loop. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

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

It is also contemplated that certain formulations can be administered orally. In an embodiment herein, the binding protein administered in this manner can be formulated with or without those carriers commonly used in the compounding of solid dosage forms (such as tablets and capsules). For example, the capsule can be designed to release the active portion of the formulation in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional reagents may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used.

Another pharmaceutical composition may contain an effective amount of binding protein mixed with non-toxic excipients suitable for the manufacture of lozenges. Solutions can be prepared in unit dose form by dissolving the lozenge in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate , Stearic acid or talc.

The additional pharmaceutical compositions herein will be obvious to those skilled in the art, which include formulations that include binding proteins in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles (eg films) or microcapsules. The sustained release matrix may include polyester, hydrogel, polylactide, copolymer of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), Ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the industry.

The pharmaceutical composition to be used for in vivo administration must generally be sterile. This can be achieved by filtration through a sterile filter membrane. In the case of freeze-drying the composition, this method can be used for sterilization before or after freeze-drying and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in solution form. In addition, the parenteral composition is usually placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (eg, lyophilized).

This document also relates to kits that include binding proteins and other reagents that can be used to detect the level of target antigens in biological samples. Such reagents can include detectable labels, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit includes a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be made of various materials such as glass or plastic. The container contains a composition effective to treat, prevent, and/or diagnose a disorder by itself or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous infusion bag or a plug that can be pierced by a hypodermic injection needle). Vial). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and/or treat the selected condition. Alternatively or additionally, the article or kit may further include a second (or third) container, the second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. It can further include other materials required from a commercial and user perspective, including other buffers, diluents, screening programs, needles and syringes.

This document also encompasses kits for producing a single dosage administration unit. The kit may each contain both a first container with dry protein and a second container with an aqueous formulation. Also included within the scope of this document are kits containing single-chamber and multi-chamber pre-filled syringes (for example, liquid syringes and lyophilized syringes).

The effective amount of the binding protein pharmaceutical composition used in treatment will depend on, for example, the treatment context and goals. Those familiar with the art will understand that the appropriate dosage level for treatment will therefore vary in part according to the following: the molecule delivered, the indication for which the binding protein is used, the route of administration, and the size of the patient (body weight, body surface, or organ size) And condition (age and general health). Therefore, clinicians can titrate the dose and change the route of administration to obtain the best therapeutic effect.

The frequency of dosing will depend on the pharmacokinetic parameters of the binding protein in the formulation used. Generally, the clinician will administer the composition until the dose to achieve the desired effect is reached. Therefore, the composition can be administered as a single dose, as two or more doses over time (they may or may not contain the same amount of the desired molecule) or as a continuous infusion via an implanted device or catheter. The further improvement of the appropriate dosage is carried out in a conventional manner by those skilled in the art, and within the scope of tasks routinely performed by those skilled in the art. The appropriate dose can be determined by using appropriate dose response data.

The route of administration of the pharmaceutical composition is consistent with known methods, for example, oral; via intravenous, intraperitoneal, intracerebral (intracerebral), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Route injection; through a sustained release system; or through an implanted device. Where necessary, the composition can be administered by bolus injection, or continuously by infusion, or by implanting a device.

The composition can also be administered locally through the implantation of a membrane, sponge or other suitable material on which the desired molecule has been adsorbed or encapsulated. In the case of an implanted device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out via diffusion, timed release bolus injection, or continuous administration.table 1. Trispecific binding protein peptide sequence molecular Peptide number (according to structural formula) SEQ ID NO sequence HER2 (WT-Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 1 1 100 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 101 3 102 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 103 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 2 1 104 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 105 3 106 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 107 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E+LC-WT-Trastuzumab)/CD28 _sup xCD3 _mid (DNAQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 8 1 108 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHDNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 109 3 110 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 111 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/56A/102S +LC-WT-Trastuzumab)/CD28supxCD3mid (32/35QQ185E) IgG4 FALA BP # 3 1 286 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 287 3 288 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 289 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28supxCD3mid (32/35QQ185E) IgG4 FALA BP # 4 1 290 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 291 3 292 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 293 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28supxCD3mid (32/35QQ185S) IgG4 FALA BP # 5 1 294 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 295 3 296 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 297 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28supxCD3mid (32/33/35QSQ185S) IgG4 FALA BP # 6 1 298 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQSAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 299 3 300 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 301 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28 _sup xCD3 _mid (32/35QQ(LC); L1 linker) IgG4 FALA BP # 9 1 112 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 113 3 114 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 115 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2-30R/55Q/102S+LC-WT-Trastuzumab/CD28supxCD3mid L1 linker IgG4 FALA BP # 10 1 116 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 117 3 118 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 119 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2-30R/56A/102S+LC-WT-Trastuzumab/ CD28supxCD3mid L1 linker IgG4 FALA BP # 11 1 120 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 121 3 122 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 123 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2-30R/56A/102E/CD28supxCD3mid L1 linker IgG4 FALA BP # 12 1 124 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 125 3 126 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 127 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2-WT+Trastuzumab/CD28supxCD3mid (32/35QQ) L1 linker IgG4 FALA BP # 15 1 128 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 129 3 130 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 131 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV DKTHT linker on HER2/CD28 _sup xCD3 _mid HC/LC) IgG4 FALA BP # 25 1 132 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 133 3 134 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 135 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLR (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 26 1 136 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 137 3 138 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 139 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 27 1 140 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 141 3 142 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 143 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLF (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 28 1 144 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 145 3 146 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 147 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV Anti-Her2/CD3/3CD28 IgG4 FALA BP # 29 1 148 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGQPKAAPDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKTKGPSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 149 3 150 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 151 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV HER230R/55Q/102E/CD28 _sup xCD3 _mid (32/33/3435 ENLR (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 31 1 152 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 153 3 154 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 155 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKESVTSGLVTESLQDSKESVTSGLVTESFYPREAKVQWKVDNALQDSKESV CD38VH1/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 1 1 156 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 157 3 158 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 159 DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTYVDNALQDSKANSTKVTSTLQSLGESFGESLGVSSQSGNSTKVTSVSSQSGN CD38hhy992/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 5' 1 160 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 161 3 162 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 163 EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIKRTVAAPSVFITKFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKDSGSLVTESFYPREAKVQWKVDNALQDSKDSGSLVTESFYPREAKVQWKVDNALQDSKDSGSLVT CD38hyb5739/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 6' 1 164 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 165 3 166 QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 167 DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLKANNFYPREAKTLSVTASVVCLLKANNFYPREAKTLSVTASVVCLLKANNFYPREAKTLSKVTASVVCLLKANNFYPREAKTLSKVTASVVCLLKANNFYPREAKTLSKVTASVVCLLKANNFYPREAKTLSKVTGSV CD38hyb6284/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 7 1 168 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 169 3 170 QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 171 DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIKTLVAAPSVFIFPPSDEQLKSGTASVVCLLGELSTYSLDYGSLVKNSVQVQVQVQTSHKSLGSLTKGTQLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLGELSTYSVGSLV CD38hhy1195/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 8 1 172 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 173 3 174 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 175 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTYVDNALQDSKANSTKVSSNRGSLGSLNNFYPREAKVQWKTYVDNALQDSKANSTKVTSTLQSLGESFGEV CD38hhy1370/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 9 1 176 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 177 3 178 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 179 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTYVDNALLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTYVDNALQDSKANSTKVSSNRGSLGESLGESLQSLGESFYPREAKVQSLGEVSSNFYPREAKVQSV CD38hu5739/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA 1 181 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 182 3 183 QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 184 DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSKANSTKGSLGSLNNFYPREAKVQWKVDNALQDSKANSTKV CD38hu6284/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA 1 185 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKDKTHTDIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKDKTHTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 186 3 187 QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 4 188 DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPDNATEQLKSGTASVVCLLNNFYPDNATEQLKSGTASVVCLLNNFYPDNATEQLKSGTASVVCLLNNFYPDNATEQLKSGTASVVCLLNNFYPDNATEQLKSG table 6. Trispecific binding protein polynucleotide sequence molecular Peptide number (according to structural formula) SEQ ID NO sequence HER2 (WT-Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 1 1 189 2 190 3 191 4 192 HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 2 1 193 2 194 3 195 4 196 HER2 (30R/55Q/102E+LC-WT-Trastuzumab)/CD28 _sup xCD3 _mid (DNAQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 8 1 197 2 198 3 199 4 200 HER2 (30R/55Q/102E +LC-WT-Trastuzumab)/CD28 _sup xCD3 _mid (32/35QQ(LC); L1 linker) IgG4 FALA BP # 9 1 201 2 202 3 203 4 204 HER2-30R/55Q/102S+LC-WT-Trastuzumab/CD28supxCD3mid L1 linker IgG4 FALA BP # 10 1 205 2 206 3 207 4 208 HER2-30R/56A/102S+LC-WT-Trastuzumab/ CD28supxCD3mid L1 linker IgG4 FALA BP # 11 1 209 2 210 3 211 4 212 HER2-30R/56A/102E/CD28supxCD3mid L1 linker IgG4 FALA BP # 12 1 213 2 214 3 215 4 216 HER2-WT+Trastuzumab/CD28supxCD3mid (32/35QQ) L1 linker IgG4 FALA BP # 15 1 217 2 218 3 219 4 220 DKTHT linker on HER2/CD28 _sup xCD3 _mid HC/LC) IgG4 FALA BP # 25 1 221 2 222 3 223 4 224 HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLR (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 26 1 225 2 226 3 227 4 228 HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLQ (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 27 1 229 2 230 3 231 4 232 HER2/ CD28 _sup xCD3 _mid (32/33/3435 ENLF (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 28 1 233 2 234 3 235 4 236 Anti-Her2/CD3/3CD28 IgG4 FALA BP # 29 1 237 2 238 3 239 4 240 HER230R/55Q/102E/CD28 _sup xCD3 _mid (32/33/3435 ENLR (LC); DKTHT linker on HC/LC) IgG4 FALA BP # 31 1 241 2 242 3 243 4 244 CD38VH1/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 1 1 245 2 246 3 247 4 248 CD38hhy992/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 5 1 249 2 250 3 251 4 252 CD38hyb5739/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 6 1 253 2 254 3 255 4 256 CD38hyb6284/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 7 1 257 2 258 3 259 4 260 CD38hhy1195/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 8 1 261 2 262 3 263 4 264 CD38hhy1370/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA BP # 9 1 265 2 266 3 267 4 268 CD38hu5739/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA 1 269 2 270 3 271 4 272 CD38hu6284/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA 1 273 2 274 3 275 4 276 Instance

The following examples illustrate the specific embodiments herein and their various uses. The examples are set forth for explanatory purposes only, and should not be considered as limiting the scope of the invention in any way. Example 1 : Development of trispecific HER2/CD28xCD3 antibody and variant anti- CD3 binding site

Immuno-oncology is a promising and emerging therapeutic method for cancer disease management. The immune system is the first line of defense against the development and progression of cancer. There is now a lot of evidence that T cells can control tumor growth in the early and late stages of the disease and prolong the survival of cancer patients. However, T cells that are specific to tumors may be restricted in many ways, which prevents them from controlling the disease.

In order to remove the restriction on uncontrolled tumor-induced T cells, a new type of antibody was developed in the form of a trispecific antibody depicted in Figure 1A to specifically activate T cells to engage cancer cells expressing HER2. These new trispecific antibodies can bind to the following three targets: HER2, CD3 and CD28. The anti-HER2 and anti-CD3 binding sites have been further optimized to achieve high-affinity binding and reduce potential production disadvantages.

HER2 amplification and overexpression can be found in molecular subtypes of breast cancer, as well as gastric cancer, ovarian cancer, lung cancer, and prostate cancer. The optimal activation of T cells requires two factors: (1) antigen recognition and (2) costimulation. In the case of using the trispecific HER2/CD28xCD3 trispecific binding protein 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 (see for example , Figure 1D ). It is believed that the trispecific antibody described in the subsequent examples recruits T cells to the tumor via HER2, and activates the engaged T cells by binding to CD3 and CD28. The resulting activation induces the killing potential of the immune cells against nearby tumor cells. Materials and methods Antibody production and characterization

Trispecific antibody variants are produced by transiently transfecting expression plastids into Expi293 cells. Five days after transfection, the supernatant from the transfected cells was collected, quantified and normalized by absorbance at 280 nm on Nano Drop. The binding of the supernatant to the corresponding antigen was determined by ELISA, and the absorbance of the parent HER2 WT tri Ab was set to 1.0. Calculate the fold change of other variants by dividing the corresponding absorbance by the absorbance of the parent Ab.

The trispecific antibody variants were purified using protein A affinity purification followed by SEC purification. The binding of the purified antibody to the corresponding antigen was determined by ELISA. The EC50 was determined based on the binding curve generated by Graphpad Prism7. result

Generate trispecific Ab variants with several mutations in the binding arms to alleviate potential production disadvantages (for example, deamidation sites). A binding ELISA analysis was performed to evaluate the binding of the indicated trispecific antibody to each of the following three targets: HER2, CD3, and CD28. In Figure IB , the HER2/CD3xCD28 trispecific antibody and the indicated anti-HER2 or anti-CD3 variant are compared with the parental trispecific antibody. The introduction of some mutation sets (for example, 32/33 QQ and 33/35QQ) into the VL domain of the anti-CD3 binding site resulted in a significant reduction in binding to CD3, while the 32/35 QQ mutation retained close to wild-type binding. MS peptide analysis showed that the binding site with DNAQ mutation in CDR-L1 (SEQ ID NO: 63) still undergoes more than 15% deamidation, while ENLQ (SEQ ID NO: 281), ENLF (SEQ ID NO : 282) and ENLR (SEQ ID NO: 283) resulted in less than 5% deamidation. Importantly, these variants also retain binding to CD3.

In addition, the binding curves of the indicated antibodies to human HER2, human CD28 and CD3 are provided in Figure 1C. The EC50 values of selected trispecific antibody variants are provided in Table E. Table E. Binding ELISA analysis of purified trispecific antibodies to determine their binding affinity to HER2, human CD3 and human CD28. Binding affinity (ELISA ) (nM ) EC50 Trispecific antibody HER2 Human CD3 Human CD28 HER2 ( WT-Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC ); DKTHT linker on HC/LC ) IgG4 FALA 162.3 566.4 1321 HER2 (30R/55Q/102E +LC-WT- Trastuzumab)/CD28supxCD3mid (32/35 QQ (LC ); DKTHT linker on HC/LC ) IgG4 FALA 93.66 364.8 871.9 HER2-30R/55Q/102E/CD28supxCD3mid (32/33/35QSQ ) DKTHT linker IgG4 FALA 83.89 3222 1024 HER2 (30R/55Q/102E+LC-WT- Trastuzumab)/CD28supxCD3mid (DNAQ (LC ); DKTHT linker on HC/LC ) IgG4 FALA 111.2 725.7 1053 HER2 (30R/55Q/102E +LC-WT- Trastuzumab)/CD28supxCD3mid (32/35QQ (LC ); L1 linker) IgG4 FALA 111.5 412.5 1345 HER230R/55Q/102E/CD28supxCD3mid (32/33/3435 ENLR (LC ); DKTHT linker on HC/LC ) IgG4 FALA 123.9 81.53 878.8 HER2 (30R/56A/102S +LC- WT-Trastuzumab)/CD28supxCD3mid (32/35QQ185E ) IgG4 FALA 516.0 5494 3631 HER2-30R/55Q/102E+LC-30Q/CD28supxCD3mid (32/35QQ ) 185S L1 linker IgG4 FALA 1540 10616 2036 HER2-30R/55Q/102E/ CD28supxCD3mid (32/33/35QSQ ) 185S L1 linker IgG4 FALA 467.0 19382 1814 HER2-30R/55Q/102E/ CD28supxCD3mid (32/33/35QSQ ) 185E L1 linker IgG4 FALA 478.6 19756 1739 DKTHT linker on HER2/CD28supxCD3mid HC/LC ) IgG4 FALA 228.9 671.2 752 HER2/CD28supxCD3mid (32/33/3435 ENLF (LC ); DKTHT linker on HC/LC ) IgG4 FALA 195.9 773.3 1466 HER2/CD28supxCD3mid (32/33/3435 ENLQ (LC ); DKTHT linker on HC/LC ) IgG4 FALA 212.1 10558 2405 HER2/CD28supxCD3mid (32/33/3435 ENLR (LC ); DKTHT linker on HC/LC ) IgG4 FALA 166.2 381.9 1051 Anti-Her2/CD3/3CD28 IgG4 FALA 176.1 516.2 870.6

Without wishing to be bound by theory, as depicted in FIG. 1D, according to / CD3 / CD28 trispecific antibodies by anti-HER2 and HER2 channel anti CD3 / CD28 T arm recruited to the cancer cells. In addition, it is believed that the engaged T cells are activated by the anti-CD28/CD3 arm. Without wishing to be bound by theory, it is believed that the killing of cancer cells occurs through T cell-mediated mechanisms (eg, perforin, granzyme). Without wishing to be bound by theory, it is considered that similar mechanisms can allow the killing of other types of tumors by replacing the antigen binding sites that recognize other tumor target proteins. Example 2 : Development of trispecific CD38/CD3xCD28 antibodies

A tri-characteristic CD38/CD3xCD28 antibody was developed and characterized for binding to CD38, CD3, and CD28 polypeptides. Materials and methods Production of CD38/CD28xCD3 trispecific antibodies

A set of anti-CD38, anti-CD3, and anti-CD28 antibodies and human IgG4 Fc domains were used to generate CD38/CD28xCD3 trispecific antibodies in the form of trispecific antibodies depicted in Figure 2A.

The trispecific binding protein is produced by transiently transfecting 4 expression plastids into Expi293 cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. To put it simply, 25% (w/w) of each plastid is diluted into Opti-MEM, mixed with the pre-diluted ExpiFectamine reagent at room temperature (RT) for 20-30 minutes, and added to Expi293 cells (2.5 x 10 ⁶ cells/ml). Transfection optimization for determining the optimal ratio of plastids is generally used to produce a trispecific binding protein with good yield and purity.

4-5 days after transfection, the supernatant was collected from the transfected cells and filtered through a 0.45 µm filter device (Nalgene). Purify the trispecific binding protein in the supernatant using a 3-step program. First, use protein A affinity purification, and use "IgG Elution Buffer" (Thermo Fisher Scientific) to elute the bound Ab. Second, the product was dialyzed against PBS (pH 7.4) overnight, and the PBS buffer was changed twice. Before the next step, filter through a 0.45 µm filter device (Nalgene) to remove any sediment. Third, use size exclusion chromatography (SEC) purification (Hiload 16/600 Superdex 200pg, or Hiload 26/600 Superdex 200pg, GE Healthcare) to remove different types of aggregates and preparations. The fractions were analyzed on reducing and non-reducing SDS-PAGE to identify the fractions containing the monomeric trispecific binding protein, and then the fractions were combined. The purified antibody can be divided into aliquots and stored at -80ºC for a long time. ELISA binding analysis

The binding affinity of the CD38/CD28xCD3 T cell adaptor to each target antigen was measured by ELISA. In simple terms, a 96-well immunoplate (Thermo Fisher Scientific) was coated with each antigen at 4ºC overnight, using 200 ng/well of each antigen in PBS (pH 7.4). The coated dish was blocked for 1 hour at RT using 5% skim milk + 2% BSA in PBS, and then washed three times with PBS + 0.25% Tween 20 (Aqua Max 400, Molecular Devices). Prepare serial dilutions of antibodies (trispecific and control Ab) and add them to the ELISA plate (100 µl/well, in duplicate), incubate at room temperature (RT) for 1 hour, and then use PBS + 0.25% Tween 20 washes 5 times. After washing, HRP-conjugated second anti-human Fab (1:5000, catalog number 109-035-097, Jackson ImmunoResearch Inc) was added to each well and incubated at RT for 30 minutes. After washing 5 times with PBS + 0.25% Tween 20, 100 µl of TMB microporous peroxidase substrate (KPL, Gaithersburg, Maryland, USA) was added to each well. The reaction was stopped by adding 50 µl of 1M H ₂ SO ₄ , and the OD450 was measured using SpectraMax M5 (Molecular Devices), and analyzed using SoftMax Pro6.3 software (Molecular Devices). Transfer the final data to GraphPad Prism software (GraphPad Software, California, USA), and draw. Use the same software to calculate EC50. Use SPR to measure trispecific antibody binding

The human CD38-His antigen (Cambridge Biologics, Cambridge, Massachusetts (MA)) was used for comprehensive kinetic analysis. The kinetic characterization of the purified antibody was performed on BIACORE 3000 (GE Healthcare) using SPR technology. Use capture analysis, which utilizes human IgG1 specific antibody capture and orientation of the antibody under study. For the capture of Fc-containing protein constructs, a human antibody capture kit (GE Healthcare) was used. For the capture of His-labeled antigen, an anti-His antibody capture kit (GE Healthcare) was used. The capture antibody was immobilized via primary amine groups (11000 RU) on the research-grade CM5 chip (GE Life Sciences) using standard procedures. The analyzed antibody is captured at a flow rate of 10 µL/min with an adjusted RU value that will result in a maximum analyte binding signal of typically 30 RU. Measure binding kinetics against trispecific antibodies. Use the analysis buffer HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at a flow rate of 30 µl/min. Regenerate the wafer surface with the regeneration solution of the corresponding capture kit. In the BIA evaluation package v4.1, the flow cell without the captured antibody is used as a reference and a 1:1 Langmuir binding model with quality transfer is used to analyze and calculate the kinetic parameters. Competitive binding analysis of daratumumab

For the darlimumab competitive binding analysis, the darlimumab amine was coupled to the active surface of the CM5 chip. The reference surface is left blank and used to subtract any non-specific binding of injected molecules. Recombinant CD38-His (Sino Biological, part number 10818-H08H) was injected on the surface of darlimumab, and then the test antibody was injected. If the monospecific anti-CD38 antibody recognizes an epitope on CD38 that is different from the epitope recognized by darlimumab, the injection of the antibody results in an increase in SPR signal. If the antibody recognizes an epitope that overlaps with darlimumab, the injection of the antibody will not increase the SPR signal. result

The binding affinity of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains was determined by SPR against human CD38. The association rate constant (K _association ), dissociation rate constant (K _dissociation ), and K _{D of the} selected trispecific antibodies are provided in Table A. The selected trispecific antibodies showed different degrees of affinity for the human CD38 antigen. Table A. Binding characteristics of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains determined by SPR against human CD38. Anti- CD38 binding domain k _association ( M ^-1 s ^-1 ) k _dissociation ( s ^-1 ) K _D ( M ) CD38VH1 5.55E+05 1.58E-03 2.85E-09 CD38hhy992 1.35E+06 1.75E-04 1.29E-10 CD38hyb6284 7.85E+05 5.12E-04 6.52E-10 CD38hyb5739 9.80E+05 5.46E-03 5.57E-09 CD38hhy1195 1.27E+06 1.80E-02 1.42E-08 CD38hhy1370 3.76E+05 3.29E-04 8.76E-10

The binding affinity of the selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with alternative anti-CD38 binding domains was then determined by ELISA as described above for human CD3, human CD28, human CD38 and cyno CD38. As shown in FIG. 2B through 2E, alternate with selected CD38 binding domain of CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies showed human CD38 (FIG. 2B), and cynomolgus monkey CD38 (FIG. 2C) Different affinities, but show similar affinities to human CD3 (Figure 2D ) and CD28 ( Figure 2E). The EC50 value was then calculated by GraphPad Prism 7.02 using a variable slope model with a four-parameter logarithmic curve. The EC50 values of the selected trispecific antibodies against human CD3, human CD28, human CD38, and cyno CD38 are provided in Table B. The control antibody is a human IgG4 isotype control. Table B. EC50 values of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains against human CD3, human CD28, human CD38 and cyno CD38. Anti- CD38 binding domain EC50 ( pM ) hCD38 Cyno CD38 hCD3 hCD28 CD38VH1 7244 2128 6742 725 CD38hhy992 229 912 33593 809 CD38hyb6284 253 290 8222 711 CD38hyb5739 984 1628 10791 825 CD38hhy1195 102537 37701 7631 697 CD38hhy1370 587 553 24968 1257

SPR competition analysis was performed to determine whether the anti-CD38 antibodies hhy6284, hhy992, hhy5379, or hhy1195 (tested as a monospecific antibody) competed with darlimumab for CD38 binding. After injecting CD38 on darlimumab (fixed on the SPR sensor chip), inject the test antibody (or darlimumab) on the darlimumab/CD38 complex. As shown in Figure 3, the injection Hyb6264, hhy992, Hyb5379 Hhy1195 and increase the SPR signal, which indicates that these antibodies recognize the CD38 and Dare adalimumab recognize different epitopes epitopes. As expected, the injection of free darlimumab (competitive binding control) did not increase the SPR signal.

The binding of anti-CD38 antibodies to human or cyno CD38 polypeptides is summarized in Table B2. Table B2. Summary of anti-CD38 binding characteristics to human or cynomolgus CD38. name ELISA huCD38 EC50 nM ELISA cynoCD38 EC50 nM FACS huCD38 EC50 nM FACS cynoCD38 EC50 nM SPR huCD38 KD M SPR cynoCD38 KD M Anti-CD38_hyb_5739 0.12 0.09 0.3 0.5 Anti-CD38_hyb_6284 0.11 0.13 0.4 0.7 Anti-CD38_hhy_992 0.09 0.08 100 288 3.65E-10 6.12E-09 Anti-CD38_hhy_1195 1.4 0.86 38 15 4.00E-08 2.60E-08

The competitive binding of the anti-CD38 antibody and darlimumab was also tested in the SPR analysis. For the daratumumab competitive binding analysis, the daratumumab amine was coupled to the active surface of the CM5 chip. The reference surface is left blank and used to subtract any non-specific binding of injected molecules. Recombinant CD38-His (Sino Biological, part number 10818-H08H) was injected on the surface of darlimumab, and then the test antibody was injected. If the antibody recognizes an epitope on CD38 that is different from the epitope recognized by darlimumab, the injection of the antibody will result in an increase in SPR signal. If the antibody recognizes an epitope that overlaps with darlimumab, the injection of the antibody will not increase the SPR signal. According to the results of these analyses, the test antibodies hhy992, hyb6284, hhy1195, and hhy1370 do not compete with draylimumab. Example 3 : The trispecific CD38/CD3xCD28 antibody promotes the lysis of human multiple myeloma and lymphoma tumor cells.

Using human multiple myeloma and lymphoma cells, an in vitro cell lysis assay was used to determine whether the trispecific CD38/CD3xCD28 antibody has anti-tumor cell activity. Materials and methods In vitro killing analysis of tumor cells using human T cells

The target tumor cells were labeled with the membrane dye PKH-26 (Sigma), and in the presence of the indicated concentration of trispecific or related control antibodies, with human PBMC or CD8 T cells enriched as effector cells at an E ratio of 10:1 : T ratio (E: T=3:1, using enriched CD8 T cells) for 24 hours. Peripheral blood mononuclear cells were isolated from normal human donors by Ficoll separation, and autologous CD8+ or whole T cells were enriched using a kit from Miltenyi Biotech (San Diego, CA). The degree of cell lysis in the target cells was determined by staining with the LIVE/DEAD™ fixable purple dead cell staining kit (Life Technologies), and by running the sample on the LSRFortessa instrument (BD Biosciences), and then using Flowjo software (Treestar ) Perform analysis and measure the number of dead cells in the marked target cell population. In Vitro Killing Analysis of Tumor Cells Using Human T Cells in the Presence of Darimumab

Dare preculture 5 nM or adalimumab labeled isotype control antibody to target PKH-26 tumor cells ⁽¹⁰⁵ cells / well) for 30 minutes, then add the indicated concentrations of TCE and trispecific human PBMC ( E: T=10: 1). After 24 hours, the degree of cell lysis in the target cells was determined by staining with the LIVE/DEAD™ fixable purple dead cell staining kit (Life Technologies), and by running the sample on the LSRFortessa instrument (BD Biosciences), and then using Flowjo software (Treestar) performs analysis and measures the number of dead cells in the marked target cell population. result

Using the human multiple myeloma cell line NCI-H929, which expresses both CD38 and CD28, the in vitro cell killing activity of the CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with alternative anti-CD38 binding domains was determined. The analysis was performed in the presence of 5 nM darlimumab or isotype control antibody (present during the analysis). As shown in FIGS. 4A-4B, in the presence and Dare adalimumab absence, trispecific antibodies are all tested in a concentration-dependent manner leading to cell lysis. Then, the EC50 values were calculated in the presence and absence of darlimumab ( Table C ). The cell-killing activity of the trispecific antibody CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA with CD38VH1 or CD38hhy1370 anti-CD38 binding domain is reduced by darlimumab, and CD38hyb5739, CD38hyb6284 or CD38hhy1195 in the presence of darlimumab The CD38 binding domain trispecific antibody exhibited a 3-8 fold reduction in cell killing activity ( Table C ). Table C. In vitro CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of darlimumab against the human multiple myeloma cell line NCI-H929 (CD38+/CD28+) in vitro Killing activity. Anti- CD38 binding domain EC50 ( pM ) CD38VH1 CD38hhy992 CD38hyb5739 CD38hyb6284 CD38hhy1195 CD38hhy1370 Darimumab is present 29.82 125.8 9.115 33.65 89.27 255.4 Human IgG1 is present 1.063 13.43 2.736 4.37 16.97 9.599

In addition, using the human lymphoma cell line OCI-LY19 that expresses CD38 but not CD28, in vitro cell lysis assays were used to measure the efficacy of selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains. Cell killing activity. The analysis was performed in the presence of 5 nM darlimumab or isotype control antibodies (which were present during the analysis). As shown in FIGS. 5A-5B in FIG., In the presence and Dare adalimumab absence, trispecific tested are all concentration-dependent manner leading to cell lysis. Then, the EC50 values were calculated in the presence and absence of darlimumab ( Table D ). The cell-killing activity of CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA with CD38VH1 anti-CD38 binding domain was reduced by about 24 times by darlimumab, and CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195, or CD38hhy1370 in the presence of darlimumab The trispecific antibodies against the CD38 binding domain also exhibited a reduction in cell killing activity ( Table D ). Table D. Selected CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with alternative anti-CD38 binding domains in the presence of darlimumab against the human lymphoma cell line OCI-LY19 (CD38+/CD28-) In vitro killing activity. Anti- CD38 binding domain EC50 ( pM ) CD38VH1 CD38hhy992 CD38hyb5739 CD38hyb6284 CD38hhy1195 CD38hhy1370 Darimumab is present 135.9 133.3 219.1 81.05 715.2 209.8 Human IgG1 is present 5.662 57.32 60.97 42.07 296.4 58.54 Example 4 : CD38/CD28xCD3 trispecific antibody promotes CMV specific immune response

As a part of adaptive immunity, T cell immunity plays a key role in controlling viral infections and cancer. It may eliminate infected cells and malignant cells, leading to the elimination of viral infections or the cure of cancer. In chronic infectious diseases such as herpes virus infection (HSV, CMV, EBV, etc.), HIV and HBV, the virus establishes its persistence in the human body through a variety of mechanisms including immunosuppression, T cell depletion, and incubation period establishment. However, virus infection usually induces virus antigen-specific immunity including antigen-specific CD8 T cells that can easily recognize infected cells for mediation by cytokine release or cytotoxic T cells (CTL). The killing process of the guide is used to control or kill. Therefore, viral antigen-specific T cell activation and/or expansion in vivo and/or ex vivo provides a therapeutic strategy against chronic viral infections.

Developed anti-CD38/CD28xCD3 trispecific antibodies and evaluated their potential in activating T cells and promoting the proliferation and/or expansion of antigen-specific T cells. These tri-specific Abs can effectively expand CD4 and CD8 effector and memory populations in vitro, including antigen-specific CD8 T central memory cells and effector memory cells. Specifically, the in vitro expansion of CMV and EBV-specific CD8 central memory cells and effector memory cells was demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibit novel characteristics by conjugating CD3/CD28/CD38 to provide signal transduction pathways that stimulate and expand T cells, which can provide treatment for chronic infectious diseases (such as HSV, CMV) , EBV, HIV-1 and HBV infection).

In this example, the ability of the CD38/CD28xCD3 trispecific antibody to promote the activation and expansion of CMV-specific T cells was determined. Materials and methods In vitro T cell proliferation measurement

The magnetic whole T cell isolation kit (Miltenyi Biotec GmbH, Germany) was used to isolate T cells from human PBMC donors by negative selection. Spread the antibody on a 96-well cell culture dish by preparing the antibody in sterile PBS and distributing 50 μL to each well (350 ng/well). The dish was then incubated at 37ºC for at least 2 hours, and then washed with sterile PBS. Add untouched T cells to an antibody-coated dish (5 x 10 ⁵ cells/mL) and incubate at 37ºC for several days. On the 4th day, the cells were subcultured onto fresh antibody-coated dishes with new cell culture medium. In some experiments using 7-day culture, only fresh medium was added without changing to a fresh antibody-coated pan. Collect cells at specific time points and use CountBright™ counting beads to count the number of cells. In vitro T cell proliferation analysis and T cell subgroup determination

Peripheral blood mononuclear cells were isolated from the blood of healthy human donors collected by Research Blood Components, LLC (Boston, Massachusetts). Add PBMC to antibody-coated plates (350 ng/well) (5 x 10 ⁵ cells/mL) as previously described above, and incubate at 37ºC for 3 and 7 days. Cells were collected at specific time points and analyzed by flow cytometry T cell subsets: naive (naïve, CCR7+ CD45RO-), Tcm (CCR7+ CD45RO+), Tem (CCR7- CD45RO+), Tregs (CD4+ Foxp3+ CD25hi). Use HLA/viral peptides restricted to PBMC donors (A*02:01/NLVPMVATV, SEQ ID NO: 284), (A*02:01/GLCTLVAML, SEQ ID NO: 285) fluorescently conjugated five Aggregate (ProImmune, Oxford, UK) detects CMV pp65-specific and EBV BMLF-specific CD8+ T cells. PBMC was obtained from HemaCare (Van Nuys, California) from a donor with known CMV or EBV infection. PMBC from a donor that was negative for restricted HLA types was used as a negative control. Dye according to the manufacturer's protocol. Quantification of CMV-specific T cells

As noted above, peripheral blood mononuclear cells (PBMC) were isolated from the blood of human donors known to be infected with CMV and added to a dish containing trispecific antibodies or control antibodies. The plate was incubated at 37ºC. Cells were collected at designated time points and analyzed by flow cytometry. result

PBMC isolated from CMV-infected human donor D ( Figure 6A to Figure 6J ) and CMV-infected human donor E ( Figure 7A to Figure 7J ) were used to test as described above with an alternative anti-CD38 binding domain ΔVH1CD38 (control ), CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195 and CD38hhy1370 CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody. In the 7-day experiment, all tested CD38 trispecific Abs activated CMV-specific T cells and promoted their proliferation with different potency and kinetics in a dose-response manner, resulting in CMV-specific CD8+ T cells (number of cells/well) (CMV donor D, Figure 6A to Figure 6B ; CMV donor E , Figure 7A to Figure 7B ). In addition, all tested CD38 trispecific Abs promoted CMV-specific central memory (T _cm ) (CMV donor D , Figure 6C to Figure 6D ; CMV donor E , Figure 7C to Figure 7D ) and effect memory (T _em ) The expansion of CD8+ T cells (CMV donor D , Fig. 6E to Fig. 6F ; CMV donor E , Fig. 7E to Fig. 7F ) (number of cells/well), the two kinds of cells were significantly expanded within 7 days. Figure 6G to Figure 6J (CMV Donor D ) and Figure 7G to Figure 7J _{(CMV Donor E) provide the CMV specific T cm} on the 0th, 3rd and 7th day in the 7-day experiment described above. And the time course of the percentage of _{T em cells.}

In summary, these data indicate that the CD38/CD28xCD3 trispecific antibody promotes CMV-specific T cells (such as CMV-specific CD8+ T cells, CMV-specific effector memory (T _em ), CD8+ T cells, and CMV-specific central memory (T _cm ) CD8+ T cells) activation and expansion. Example 5 : CD38/CD28xCD3 trispecific antibody promotes EBV specific immune response

Next, the ability of the CD38/CD28xCD3 trispecific antibody to promote the activation and expansion of Estein-Barr virus (EBV) specific T cells was determined. Materials and methods Quantification of EBV-specific T cells

As noted above, peripheral blood mononuclear cells (PBMC) were isolated from the blood of human donors known to be infected with EBV and added to a dish containing trispecific antibodies or control antibodies. The plates are incubated at 37ºC for up to 11 days. Cells were collected at designated time points and analyzed by flow cytometry. result

PBMC isolated from EBV-infected donor C ( Fig. 8A to Fig. 8J ) and EBV-infected donor D ( Fig. 9A to Fig. 12 ) were also used to test with alternative anti-CD38 binding domain ΔVH1CD38 (control) as described above , CD38VH1, CD38hhy992, CD38hyb5739, CD38hyb6284, CD38hhy1195 and CD38hhy1370 CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody. In the 7-day experiment, all tested CD38 trispecific Abs activated T cells with different potency and kinetics in a dose-response manner and promoted the proliferation of EBV-specific T cells, resulting in an increase in EBV-specific CD8+ T cells (cell Number/hole) (EBV donor C, Figure 8A to Figure 8B ; EBV donor D , Figure 9A to Figure 9B ). In addition, all tested CD38 tri-specific Abs promoted EBV-specific central memory (T _cm ) (EBV donor C , Figure 8C to Figure 8D ; EBV donor D , Figure 9C to Figure 9D ) and effect memory (T _em ) The expansion (number of cells/well) of CD8+ T cells (EBV donor C , Fig. 8E to Fig. 8F ; EBV donor D , Fig. 9E to Fig. 9F ), the two kinds of cells were significantly expanded within 7 days. Figures 8G to 8J (EBV donor C ) and Figures 9G to 12 _{(EBV donor D) provide the EBV specific T cm} shown in the 7 day experiment described above on the 0th, 3rd and 7th days And the time course of the percentage of _{T em cells.}

In summary, these data indicate that the CD38/CD28xCD3 trispecific antibody promotes EBV-specific T cells (such as EBV-specific CD8+ T cells, EBV-specific effector memory (T _em ), CD8+ T cells, and EBV-specific central memory (T _cm ) CD8+ T cells) activation and expansion. Example 6 : Anti-tumor effect of Her2/CD28 x CD3 trispecific antibody in tumor-bearing mice

In this example, the anti-tumor effect of the Her2/CD28 x CD3 trispecific antibody was tested in a ZR-75-1 tumor-bearing Nod scidγ (NSG) mouse model transplanted with in vitro expanded T cells. Materials and methods

The NSG mice were divided into 5 groups, each with 10 mice. On day 0, ZR-75-1 human breast cancer cells were implanted into the mammary fat pad of each mouse at a rate of 5 million cells/mouse with 50% Matrigel. On day 17/18, the expansion of human CD3+ T cells began. Randomization of mice was performed on day 24 when the tumor was approximately 150 mm ^3. On the 25th day, all mice were transplanted with 300 μL/mouse with 10 million cells of human CD3+ T cells expanded in vitro (1QW, 1 IP injection).

Starting from day 25, a group of mice received a single vehicle dose (8% w/v sucrose, 0.05% w/v polysorbate 80, 10 mM histidine, pH 5.5), while the other 4 groups The mice received Her2/CD28 x CD3 trispecific antibodies, both of which were 10 mL/kg. The group receiving trispecific antibodies was dosed at 100, 10, 1, or 0.1 μg/kg. The antibody or vehicle was administered 1 QW intravenously in 2 doses (eg, 25 and 32 days). Blood and tumor tissue were collected on the 38th or 39th day. result

The Her2/CD28 x CD3 trispecific antibody (binding protein #2 from Table 1, corresponding to SEQ ID No: 104-107) and vehicle control were directed against NSG mice transplanted with human T cells expanded in vitro as described above The effects on human breast tumor growth were compared in the model. Treatment with the highest dose (100 ug/kg) of Her2/CD28 x CD3 trispecific antibody resulted in the most significant tumor growth inhibition and regression, but the 10 ug/kg dose also showed anti-tumor effects ( Figure 13A and Figure 13D ) . No significant weight loss was observed ( Figure 13B ). The individual tumor volume over time for each trispecific antibody treatment group is provided in Figure 13C .

Next, the effect of trispecific antibody therapy on individual immune cell subgroups was examined. Flow cytometry was used to measure human CD45+, human CD8+ and human CD4+ cell populations and mouse CD45+ cells ( Figure 14A to Figure 14C ). The highest dose (100 ug/kg) of the trispecific antibody caused the depletion of human CD4+ cells, and this effect was dose-dependent ( Figure 14B and Figure 14C ). The count of human CD8+ cells is largely unaffected by the administration of trispecific antibodies.

The effect of trispecific antibody treatment on tumor infiltrating lymphocytes (TIL) was also evaluated by immunohistochemical (IHC) staining against human CD45, CD4 and CD8. Using H&E staining, tumors from the low-dose group (1 ug/kg or 0.1 ug/kg trispecific antibody) had roughly the same size as the vehicle control group. As shown in FIGS. 15A to 15C, the Her2 / CD28 x CD3 antibodies specific three groups receiving the low dose, the increase in human TIL, but in the high dose group, the human TIL are rare. IHC images were also quantitatively checked ( Figure 16A to Figure 16C ). These results indicate that CD45+ and CD8+ cells were significantly reduced in the higher trispecific antibody dose groups (100 ug/kg and 10 ug/kg).

Compared with vehicle control, tumors from the high-dose trispecific antibody treatment group (100 ug/kg or 10 ug/kg) were characterized by scarce TIL. Moderate to large amounts of CD45+, CD4+ or CD8+ human TIL were observed in the 1 ug/kg and 0.1 ug/kg trispecific antibody treatment groups. These TILs are mainly present at the edge of the tumor, but occasionally extend deeper into the core of the tumor.

In summary, these results confirm that the use of 2 intravenous doses of HER2 targeting HER2 and T cell-conjugating trispecific antibodies at 100 ug/kg or 10 ug/kg to treat ZR-75-1-carrying breasts transplanted with activated T cells in vitro NSG mice with tumors resulted in a significant reduction in tumor volume and at the same time a significant reduction in TIL. Compared with the vehicle control, at the 1 ug/kg trispecific antibody dose, the trend of TIL increase is small and inconsistent. Example 7 : The anti- HER2 and anti- CD3 antigen binding domain sequences in the Her2/CD28 x CD3 trispecific antibody kill cancer cells

This example describes the killing effect of anti-Her2 and anti-CD3 variable domain sequences on target cells. In this example, a Her2/CD28 x CD3 trispecific with a wild-type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without 32/35 QQ mutations in the VL domain (see Example 1) Antibody ("control") and Her2/CD28 x CD3 trispecific antibody #1-6 from Table 1 (corresponding to SEQ ID No: 100-103, 104-107, 286-289, 290-293, 294, respectively -297 and 298-301) for comparison. Materials and methods

A magnetic bead separation kit (Miltenyi Biotec) was used to isolate CD8+ T cells from human PBMCs from healthy donors. T cells are used as effector cells for breast cancer cell lines expressing various levels of HER2 at a ratio of 3:1 (effector: target). The cells were incubated with experimental or control trispecific antibodies for 2 days, and then collected by flow cytometry using viability dye (Invitrogen) and PKH26 target cell staining (Sigma). For each trispecific Ab, the average EC50 of target cell lysis was calculated based on 2-3 PBMC donors. result

In vitro cell lysis against the following three HER2+ breast cancer target cell lines characterized all trispecific antibodies: HCC1954, BT20, and MDA-MB-231. As assessed by flow cytometry (up to about 150,000 receptors/cell), IHC (3+) or HercepTest HER2 performance analysis (3+), it was found that HCC1954 breast cancer cells showed high levels of HER2 ( Figure 17A ). As assessed by flow cytometry (approximately 60,000 receptors/cell), IHC (1+), or HercepTest HER2 performance analysis (1+), it was found that BT20 breast cancer cells exhibited intermediate levels of HER2 ( Figure 17C ). As assessed by flow cytometry (approximately 9,000 receptors/cell), IHC (0+) or HercepTest HER2 performance analysis (0), it was found that MDA-MD-231 breast cancer cells showed low levels of HER2 ( Figure 17E ) . The results of the cell killing analysis targeting HCC1954, BT20 or MDA-MB-231 are shown in Figure 17B , Figure 17D, and Figure 17F , respectively. Binding protein #2 and control, or binding protein #1 and #5 and Contrast. The results confirmed that the Her2/CD28 x CD3 trispecific antibody with the 30R/55Q/102E mutation in the anti-HER2 arm and the 32/35QQ mutation in the VL domain of the anti-CD3 arm showed high resistance against all three cell lines. Improved target cell killing, especially at lower antibody concentrations.

Determined the average in vitro cell killing of all trispecific antibodies targeting the above three breast cancer cell lines (HCC1954, BT20 and MDA-MB-231) and gastric cancer cell lines OE19 (high HER2 performance) and GSU (medium HER2 performance) EC50 (pM). In general, Her2/CD28 x CD3 trispecific antibodies with mutations in the VL domain of the anti-HER2 arm and the anti-CD3 arm were shown to be directed against all three breast cancer cell lines ( Figure 18A ) and two gastric cancer cell lines ( Figure 18B ) lower EC50 (and therefore excellent cell killing). These results confirm that although all trispecific antibodies can induce cell killing on HER2+ cells, the mutant trispecific antibodies consistently show improved cell killing efficacy against multiple target cell types.

no

Figure 1A provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that bind to the following three target proteins: CD28, CD3 and HER2. The first pair of polypeptides have dual variable domains (VH1-VH2 and VL2-VL1) with staggered orientations, which form two antigen binding sites that recognize CD3 and CD28, and the second pair of polypeptides have Single variable domain (VH3 and VL3), which forms a single antigen binding site that recognizes HER2. The trispecific binding protein shown in Figure 1A uses a constant region with a "knob-into-hole" mutation, where the knob is located on a second pair of polypeptides with a single variable domain.

Figure IB provides the fold change in binding affinity (relative to the parent) of the anti-CD28/CD3/HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. The mutation from 3233QQ to QEQ (top-down) refers to the mutation (indicated by *) introduced in residues 32 to 35 of the VL domain of the anti-CD3 binding site; the remaining mutations are introduced into trastuzumab ) In the VH or VL domain of the anti-HER2 binding site (indicated by #; numbering according to Kabat). For mutations in the anti-HER2 binding site, mutation 30Q was introduced into the VL domain, and the remaining mutations were introduced into the VH domain. The binding affinity was measured by ELISA, and the value provided is relative to the parental trispecific antibody.

Figure 1C provides the binding curves of the binding of the indicated trispecific antibodies to human HER2, human CD28 and CD3, as determined by ELISA.

Figure ID provides a proposed mechanism of action for HER2/CD28xCD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing.

Figure 2A provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that bind to the following three target proteins: CD28, CD3, and CD38. The first pair of polypeptides have dual variable domains (VH1-VH2 and VL2-VL1) with staggered orientations, which form two antigen binding sites that recognize CD3 and CD28, and the second pair of polypeptides have Single variable domains (VH3 and VL3), which form a single antigen binding site that recognizes CD38. The trispecific binding protein shown in Figure 2A uses an IgG4 constant region with a "knob and socket structure" mutation, where the knob is located on a second pair of polypeptides with a single variable domain.

Figures 2B to 2E show the CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domain against the target antigen human CD38 ( Figure 2B ) and cynomolgus CD38 (Figure 2B) as measured by ELISA. 2C ), the binding affinity of human CD3 ( Figure 2D ) and human CD28 ( Figure 2E).

Figure 3 shows the SPR competition analysis of darlimumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains binding to CD38. If the antibody recognizes an epitope on CD38 that is different from the epitope recognized by darlimumab, the injection of the antibody results in an increase in SPR signal. If the antibody recognizes an epitope that overlaps with darlimumab, the injection of the antibody will not increase the SPR signal.

Figures 4A to 4B show the in vitro cell killing activity of the CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domain against human multiple myeloma NCI-H929 cells (CD38+/CD28+). The analysis was performed in the presence of 5 nM isotype control antibody ( Figure 4A ) or darlimumab ( Figure 4B ). In the presence of darlimumab, the trispecific antibody continues to exhibit cell-killing activity.

Figures 5A to 5B show the in vitro cell killing activity of the CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domain against human lymphoma OCI-Ly19 cells (CD38+/CD28-). The analysis was performed in the presence of 5 nM isotype control antibody ( Figure 5A ) or darlimumab ( Figure 5B ). Darlimumab caused a decrease in the cell killing activity of the anti-CD38/CD28xCD3 trispecific antibody.

Figures 6A to 6J show the characterization of in vitro T cell subgroup expansion in PBMC collected from CMV-infected donor D, and this in vitro T cell subgroup expansion line responds to CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA A trispecific antibody with the indicated alternative anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1/ΔCD28sup x ΔCD3mid IgG4 FALA). The T cell population was measured at the indicated time point (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Use flow cytometry to quantify CMV-specific CD8+ T cells ( Figure 6A to Figure 6B ), CMV-specific T _cm CD8+ cells ( Figure 6C to Figure 6D ), and CMV-specific T _em CD8+ cells ( Figure 6E to Figure 6F) ). _{In addition, the percentages of CMV-specific T cm} ( Figure 6G to Figure 6H ) and T _em ( Figure 6I to Figure 6J ) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in a dose-response manner.

Figures 7A to 7J show the characterization of the in vitro T cell subgroup expansion in PBMC, the PBMC line was collected from CMV-infected donor E, and the in vitro T cell subgroup expansion line responded to CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody, the trispecific antibody system has the indicated anti-CD38 binding domain, as a negative control (ΔCD38VH1/ΔCD28sup x ΔCD3mid IgG4 FALA). The resistance system shown from top to bottom in the legend is shown from left to right in the figure. The T cell population was measured at the indicated time point (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM. Use flow cytometry to quantify CMV-specific CD8+ T cells ( Figure 7A to Figure 7B ), CMV-specific T _cm CD8+ cells ( Figure 7C to Figure 7D ), and CMV-specific T _em CD8+ cells ( Figure 7E to Figure 7F) ). _{In addition, the percentages of CMV-specific T cm} ( Figure 7G to Figure 7H ) and T _em ( Figure 7I to Figure 7J ) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in a dose-response manner.

Figures 8A to 8J show the characterization of in vitro T cell subgroup expansion in PBMC collected from EBV-infected donor C, and this in vitro T cell subgroup expansion line responds to CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA A trispecific antibody with the indicated alternative anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1/ΔCD28sup x ΔCD3mid IgG4 FALA). The T cell population was measured at the indicated time point (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Use flow cytometry to quantify EBV-specific CD8 + T cells ( Figure 8A to Figure 8B ), CMV-specific T _cm CD8+ cells ( Figure 8C to Figure 8D ), and CMV-specific T _em CD8+ cells ( Figure 8E to Figure 8F) ). _{In addition, the percentages of EBV-specific Tcm} ( Figure 8G to Figure 8H ) and _Tem ( Figure 8I to Figure 8J ) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in a dose-response manner.

Figures 9A to 12 show the characterization of in vitro T cell subgroup expansion in PBMC collected from EBV-infected donor D, and this in vitro T cell subgroup expansion line responds to CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA A trispecific antibody with the indicated alternative anti-CD38 binding domain. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1/ΔCD28sup x ΔCD3mid IgG4 FALA). The T cell population was measured at the indicated time point (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Use flow cytometry to quantify EBV-specific CD8 + T cells ( Figure 9A to Figure 9B ), EBV-specific T _cm CD8 + cells ( Figure 9C to Figure 9D ), and EBV-specific T _em CD8 + cells ( Figure 9E to Figure 9F) ). _{In addition, the percentages of EBV-specific T cm} ( Figure 9G to Figure 10 ) and T _em ( Figure 11 to Figure 12 ) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells with different potency and kinetics in a dose-response manner.

Figures 13A to 13D show the time (days) changes of tumor volume (Figure 13A ) and body weight ( Figure 13B ) in NSG mice transplanted with human CD3+ T cells expanded in vitro and carrying ZR-75-1 tumors . A group of 10 mice was treated with vehicle or Her2/CD28 x CD3 trispecific antibody at the indicated dose. The arrow indicates the number of days cast. Tumor volume is depicted as mean ± SEM, mm ³ . Changes in body weight are depicted as% change, mean ± SEM. The X axis shows the number of days after implantation of ZR-75-1 cells. ^{The tumor volume (mm 3} ) of individual mice in each treatment group over time is shown in Figure 13C . The tumor weight (mg) for each treatment group is shown in Figure 13D . ** = p <0.001; *** = p <0.0003 (two-way ANOVA, control relative to 100 and 10 ug/kg).

Figures 14A to 14C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on T cells from whole blood. Figure 14A shows the analysis of hCD45+, CD8+, CD4+ and mCD45+ cells by flow cytometry. Figure 14B shows the effect of control or Her2/CD28 x CD3 trispecific antibody treatment (at the indicated dose) on hCD45+, CD8+, CD4+ and mCD45+ cell counts. Figure 14C shows the effect of control or Her2/CD28 x CD3 trispecific antibody treatment (at the indicated dose) on the human cell ratio (CD4+/CD45+ and CD8+/CD45+). For each x-axis parameter shown in Figure 14B and Figure 14C , the conditions are (from left to right): control, 100 ug/kg trispecific antibody, 10 ug/kg trispecific antibody, 1 ug/kg trispecific Specific antibodies and 0.1 ug/kg trispecific antibodies. The percentages shown in Figure 14B and Figure 14C are based on the control sample relative to 100 ug/kg.

Figures 15A to 15C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TIL) as examined by immunohistochemistry (IHC). The arrow indicates the tumor-infiltrating T cells identified in the ZR-75-1 breast tumor. The upper picture is 1X magnification; the lower picture is 20X magnification. In the two sets of images, the staining of human CD45, human CD4, and human CD8 are shown from left to right. Shown are tumors from mice treated with vehicle control ( Figure 15A ), 100 ug/kg trispecific antibody ( Figure 15B ), or 0.1 ug/kg trispecific antibody ( Figure 15C).

Figures 16A to 16C show a quantitative analysis of the effect of Her2/CD28 x CD3 trispecific antibody treatment on TIL as measured by IHC. Each point represents a tumor from a single mouse; rectangles represent group averages; and error bars indicate standard deviations. * = p<0.05, compared with vehicle control group (ANOVA). Shows the number of CD45+ (Figure 16A ), CD4+ (Figure 16B ) or CD8+ (Figure 16C ) cells. In Figure 16C , because there are too many non-specific signals in the CD8 IHC slide, the $area quantification method is used instead of the cell counting algorithm for CD8+ cells.

Figures 17A to 17F show that compared with the Her2/CD28 x CD3 trispecific antibody with mutations in the VL domains of the anti-HER2 arm and anti-CD3 arm (numbered as shown in Table 1), it has wild-type trastole Bezumab antigen binding domain and Her2/CD28 x CD3 trispecific antibody against CD3 antigen binding domain without 32/35 QQ mutations in the VL domain ("ctl"), in the presence of human CD8+ T cells In vitro cell lysis of HER2+ breast cancer target cells. Depicted are cell-killing activities against cell lines with different HER2 performance: HCC1954 with high HER2 performance ( Figure 17A ), BT20 with medium HER2 performance ( Figure 17C ) and MDA-MD-231 with low HER2 performance ( Figure 17E ) . Shown is a graph depicting the cell killing of target cells HCC1954 ( Figure 17B ), BT20 ( Figure 17D ) and MDA-MD-231 ( Figure 17F ) as a function of antibody concentration, which compares binding protein #2 and ctl, Or compare binding proteins #1 and #5 with ctl.

Figures 18A and 18B summarize the average EC50 (pM) of in vitro cell killing of the experimental or control Her2/CD28 x CD3 trispecific antibody against the indicated breast cancer ( Figure 18A ) or gastric cancer cell line ( Figure 18B). The amino acid sequences of the indicated trispecific antibodies are provided in Table 1.

Claims (89)

A binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [ I] and the second polypeptide chain comprises the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and the third polypeptide chain comprises the following The structure represented by the formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain includes the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the first Immunoglobulin light chain variable domain; _VL2 is the second immunoglobulin light chain variable domain; _VL3 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; _CL is the immunoglobulin light chain constant domain; C _H1 is C _H1 immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; hinge and C _H2 C _H1 is connected to the structure And L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; And where V _H1 and V _L1 form a first antigen binding site; where V _H2 and V _L2 form a second antigen binding site that binds to a CD3 polypeptide, and wherein the V _H2 domain comprises: GFTFTKAW (SEQ ID NO: 55) ) The CDR-H1 sequence of the amino acid sequence, the CDR-H2 sequence containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) and the CDR-H3 containing the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57) Sequence, and the V _L2 domain comprises: _{QSLVHX 1} NX ₂ X ₃ TY (SEQ ID NO: 180) _{containing X 1} is E or Q, X ₂ is A or L and X ₃ is Q, R or F The CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), the CDR-L2 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64), and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65) ; And where V _H3 and V _L3 form a third antigen binding site. The binding protein according to claim 1, wherein the first binding site binds a CD28 polypeptide. The binding protein according to claim 2, wherein the V _H1 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), an amine containing IYPGNVNT (SEQ ID NO: 50) CDR-H2 sequence comprising amino acid sequence and TRSHYGLDWNFDV (SEQ ID NO: 51) CDR-H3 amino acid sequences of the sequence; V _L1 and said domain comprises: comprising QNIYVW (SEQ ID NO: 52) amine The CDR-L1 sequence of the acid sequence, the CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53), and the CDR-L3 sequence containing the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). The requested item binding protein of claim 3, wherein said V _H1 domain comprises QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) The amino acid sequence and / or the V _L1 domain comprises DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92) The amino acid sequence. The requested item according to any one of claim 4-1 binding protein, wherein the sequence of the CDR-L1 V _L2 domain comprising the amino acid sequence selected from the following: QSLVHQNAQTY (SEQ ID NO: 59 ), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61) and QSLVHENLRTY (SEQ ID NO: 62). Item 5 The binding protein according to the request, wherein the domain comprises a V _H2 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302) amino acid sequences, and / or V _L2 domain comprising the amino acid sequence selected from: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). The binding protein according to any one of claims 1 to 6, wherein the third antigen binding site binds to a tumor target protein. The binding protein according to any one of claims 1 to 6, wherein the third antigen binding site binds to a human CD38 polypeptide. The binding protein according to claim 8, wherein: (a) the V _H3 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), and containing IYPGQGGT (SEQ ID NO: CDR-H2 sequence 14) and comprising the amino acid sequence ARTGGLRRAYFTY (SEQ ID NO: 15) the amino acid sequence of CDR-H3 sequence; V _L3 and said domain comprises: comprising QSVSSYGQGF (SEQ ID NO: 16 ) CDR-L1 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17), CDR-L2 containing the amino acid sequence of GAS (SEQ ID NO: 17), and CDR-L3 containing the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18) Sequence; (b) The V _H3 domain contains: CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), CDR-H1 containing the amino acid sequence of FDPEDGET (SEQ ID NO: 20) and H2 sequence comprising TTGRFFDWF (SEQ ID NO: 21) CDR-H3 amino acid sequences of the sequence; V _L3 and said domain comprises: comprising QSVISRF (SEQ ID NO: 22) the amino acid sequence of CDR-L1 Sequence, CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23) and CDR-L3 sequence containing the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24); (c) the V _H3 structure The domain contains: the CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), the CDR-H2 sequence containing the amino acid sequence of INPGSGST (SEQ ID NO: 26), and the CDR-H2 sequence containing the amino acid sequence of ARYAYGY (SEQ ID NO: 27) the amino acid sequence of CDR-H3 sequence; V _L3 and said domain comprises: comprising QNVGTA (SEQ ID NO: 28) amino acid sequences of CDR-L1 sequence, comprising SAS (SEQ ID NO: 29 ) The CDR-L2 sequence of the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30) and the CDR-L3 sequence of the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30); (d) The V _H3 domain includes: GYSFTNYA (SEQ ID NO: 31) the CDR-H1 sequence of the amino acid sequence, the CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32) and the CDR-H2 sequence containing the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33) H3 sequence; and the _VL3 domain contains: The CDR-L1 sequence of the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), the CDR-L2 sequence of the amino acid sequence of KVS (SEQ ID NO: 35), and the amine containing SQSTHVPLT (SEQ ID NO: 36) (E) The V _H3 domain includes: the CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), the CDR-H1 sequence containing IWYDGSNK (SEQ ID NO: 38) CDR-H2 sequence comprising the amino acid sequence and ARDPGLRYFDGGMDV (SEQ ID NO: 39) CDR-H3 amino acid sequences of the sequence; V _L3 and said domain comprises: comprising QGISSY (SEQ ID NO: 40) amine CDR-L1 sequence of the base acid sequence, CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41), and CDR-L3 sequence containing the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42); or (f) The V _H3 domain includes: a CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), and a CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 44) and comprising ARMFRGAFDY (SEQ ID NO: 45) CDR-H3 amino acid sequences of the sequence; V _L3 and said domain comprises: comprising QGIRND (SEQ ID NO: 46) amino acid sequences of CDR-L1 sequence, The CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47) and the CDR-L3 sequence containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). The requested item binding protein of claim 9, wherein: (a) the V _H3 domain comprises QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79) The amino acid sequence and / or the V _L3 domain comprises DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80) the amino acid sequences; (b) the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81 amino acid sequence) of, and / or the V _L3 domain comprises EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82) the amino acid sequence; (c) the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83 amino acid sequence) of, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84) (D) The V _H3 domain contains QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTM TVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85) The amino acid sequence and / or the V _L3 domain comprises DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86) amino acid sequences; (e) the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS ( SEQ ID NO: 87) the amino acid sequence and / or the V _L3 domain comprises DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88) the amino acid sequence; or (f) the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89) the amino acid sequence and / or the V _L3 domain comprises AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90) the amino acid sequence. The binding protein according to claim 8, wherein: (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid of SEQ ID NO: 158 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 159 or the amino acid sequence of SEQ ID NO: 159 The amino acid sequence is at least 95% identical to the amino acid sequence; (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid of SEQ ID NO: 162 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 163 or the amino acid sequence of SEQ ID NO: 163 The amino acid sequence is at least 95% identical to the amino acid sequence; (c) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid of SEQ ID NO: 166 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 167 or the amino acid sequence of SEQ ID NO: 167 The amino acid sequence is at least 95% identical to the amino acid sequence; (d) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid of SEQ ID NO: 170 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 171 or the amino acid sequence of SEQ ID NO: 171 The amino acid sequence is at least 95% identical to the amino acid sequence; (e) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid of SEQ ID NO: 174 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 175 or the amino acid sequence of SEQ ID NO: 175 The amino acid sequence is at least 95% identical to the amino acid sequence; (f) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid of SEQ ID NO: 178 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 179 or the amino acid sequence of SEQ ID NO: 179 The amino acid sequence is at least 95% identical to the amino acid sequence; (g) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid of SEQ ID NO: 183 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 184 or the amino acid sequence of SEQ ID NO: 184 The amino acid sequence is at least 95% identical to the amino acid sequence; or (h) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid of SEQ ID NO: 187 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 188 or the amino acid sequence of SEQ ID NO: 188 The amino acid sequence is at least 95% identical to the amino acid sequence. The binding protein according to any one of claims 1 to 7, wherein the third antigen binding site binds to a human HER2 polypeptide. The binding protein according to claim 12, wherein: (a) the V _H3 domain comprises: CDR-H1 containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2) Sequence, 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 SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7) or SRWGGSGFYAMDY (SEQ ID NO: 8) the amino acid sequence of CDR-H3 sequence; V _L3 and said domain comprises: comprising QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10) CDR-L1 sequence of the amino acid sequence, CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and amino acid sequence containing QQHYTTP (SEQ ID NO: 12) (B) The V _H3 domain contains: the CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), and the amino acid containing IYPTNGYT (SEQ ID NO: 3) CDR-H2 sequence comprising the sequence and SRWGGDGFYAMDY (SEQ ID NO: 6) the amino acid sequence of the CDR-H3 sequence; V _L3 and said domain comprises: comprising QDVNTA (SEQ ID NO: 9) amino acid sequence of The CDR-L1 sequence, the CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 11), and the CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (c) The V _H3 domain includes: the CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), the CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and the CDR-H2 sequence containing the amino acid sequence of SRWGGEGFYAMDY ( SEQ ID NO: CDR-H3 sequence 7) of the amino acid sequence; V _L3 and said domain comprises: comprising QDVNTA (SEQ ID NO: 9) amino acid sequences of CDR-L1 sequence, comprising SAS (SEQ ID NO: 11) the CDR-L2 sequence of the amino acid sequence and the CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (d) The V _H3 domain contains: GFNIRDT The CDR-H1 sequence of the amino acid sequence of Y (SEQ ID NO: 2), the CDR-H2 sequence containing the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and the amine containing SRWGGSGFYAMDY (SEQ ID NO: 8) CDR-H3 sequence acid sequence; V _L3 and said domain comprises: comprising QDVNTA (SEQ ID NO: 9) amino acid sequences of CDR-L1 sequence, comprising SAS (SEQ ID NO: 11) amine The CDR-L2 sequence of the acid sequence and the CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12); (e) The V _H3 domain contains: an amine containing GFNIRDTY (SEQ ID NO: 2) The CDR-H1 sequence of the base acid sequence, the CDR-H2 sequence containing the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and the CDR-H3 sequence containing the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and the V _L3 domain comprises: comprising QDVNTA (SEQ ID NO: 9) CDR-L1 sequence of the amino acid sequence, comprising SAS: CDR-L2 sequences (SEQ ID NO 11) and comprising the amino acid sequence QQHYTTP (SEQ ID NO: 12) the CDR-L3 sequence of the amino acid sequence; (f) The V _H3 domain includes: the CDR-H1 sequence containing the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), CDR-H2 sequence: (5 SEQ ID NO) of the amino acid sequence containing SRWGGEGFYAMDY (SEQ ID NO: 7) IYPTNAYT the amino acid sequence of the CDR-H3 sequence; V _L3 and said domain comprises: comprising QDVNTA The CDR-L1 sequence of the amino acid sequence of (SEQ ID NO: 9), the CDR-L2 sequence of the amino acid sequence of SAS (SEQ ID NO: 11), and the amino group of QQHYTTP (SEQ ID NO: 12) The CDR-L3 sequence of the acid sequence; or (g) the V _H3 domain comprises: the CDR-H1 sequence containing the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), and the sequence containing IYPTNGYT (SEQ ID NO: 3) CDR-H2 sequence comprising the amino acid sequence and SRWGGDGFYAMDY (SEQ ID NO: 6) the amino acid sequence of the CDR-H3 sequence; V _L3 and said domain comprises: comprising QDVQTA (SEQ ID NO: 10) amine Base acid sequence of CDR-L1 sequence, containing SAS (SEQ ID NO: 11) The CDR-L2 sequence of the amino acid sequence and the CDR-L3 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). The requested item binding protein of claim 13, wherein: (a) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) , EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78) of Amino acid sequence; (b) the V _H3 domain package Containing EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) amino acid sequences, and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) amino acid sequence; (c) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) the amino acid sequence; (d) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) the amino acid sequence; (e) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74) The amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) The amino acid sequences; (f) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS ( SEQ ID NO: 76) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) the amino acid sequence; or (g) the V _H3 domain comprises EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72) the amino acid sequence and / or the V _L3 domain comprises DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78) the amino acid sequence. The binding protein according to claim 12, wherein: (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid of SEQ ID NO: 102 Sequence or an amino acid sequence that is at least 95% identical to 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 the amino acid sequence of SEQ ID NO: 103 The amino acid sequence is at least 95% identical to the amino acid sequence; (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid of SEQ ID NO: 106 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 107 or the amino acid sequence of SEQ ID NO: 107 The amino acid sequence is at least 95% identical to the amino acid sequence; (c) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid of SEQ ID NO: 114 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 115 or the amino acid sequence of SEQ ID NO: 115 The amino acid sequence is at least 95% identical to the amino acid sequence; (d) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid of SEQ ID NO: 118 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 119 or the amino acid sequence of SEQ ID NO: 119 The amino acid sequence is at least 95% identical to the amino acid sequence; (e) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid of SEQ ID NO: 122 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 123 or the amino acid sequence of SEQ ID NO: 123 The amino acid sequence is at least 95% identical to the amino acid sequence; (f) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid of SEQ ID NO: 126 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 127 The amino acid sequence is at least 95% identical to the amino acid sequence; (g) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain Comprising the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid of SEQ ID NO: 130 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 131 or the amino acid sequence of SEQ ID NO: 131 The amino acid sequence is at least 95% identical to the amino acid sequence; (h) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid of SEQ ID NO: 134 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 135 or the amino acid sequence of SEQ ID NO: 135 The amino acid sequence is at least 95% identical to the amino acid sequence; (i) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid of SEQ ID NO: 138 Sequence or an amino acid sequence that is at least 95% identical to 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 the amino acid sequence of SEQ ID NO: 139 The amino acid sequence is at least 95% identical to the amino acid sequence; (j) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid of SEQ ID NO: 142 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 143 or the amino acid sequence of SEQ ID NO: 143 The amino acid sequence is at least 95% identical to the amino acid sequence; (k) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid of SEQ ID NO: 146 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 147 or the amino acid sequence of SEQ ID NO: 147 The amino acid sequence is at least 95% identical to the amino acid sequence; (1) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid of SEQ ID NO: 150 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence of SEQ ID NO: 151 The amino acid sequence is at least 95% identical to the amino acid sequence; (m) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid of SEQ ID NO: 154 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 155 or the amino acid sequence of SEQ ID NO: 155 The amino acid sequence is at least 95% identical to the amino acid sequence; (n) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 286; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 287; the third polypeptide chain comprises the amino acid of SEQ ID NO: 288 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 288; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 289 or the amino acid sequence of SEQ ID NO: 289 The amino acid sequence is at least 95% identical to the amino acid sequence; (o) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 290; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 291; the third polypeptide chain comprises the amino acid of SEQ ID NO: 292 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 292; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 293 or the amino acid sequence of SEQ ID NO: 293 The amino acid sequence is at least 95% identical to the amino acid sequence; (p) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 294; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 295; the third polypeptide chain comprises the amino acid of SEQ ID NO: 296 Sequence or an amino acid sequence that is at least 95% identical to 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 the amino acid sequence of SEQ ID NO: 297 The amino acid sequence is at least 95% identical to the amino acid sequence; or (q) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 298; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 299; the third polypeptide chain comprises the amino acid of SEQ ID NO: 300 Sequence or an amino acid sequence that is at least 95% identical to 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 the amino acid sequence of SEQ ID NO: 301 The amino acid sequence is at least 95% identical to the amino acid sequence. The binding protein according to any one of claims 1 to 15, wherein _{at least one of L 1} , L ₂ , L ₃ or L ₄ is independently 0 amino acids in length. The binding protein according to any one of claims 1 to 15, wherein (a) L ₁ , L ₂ , L ₃ and L ₄ are each independently the length of zero amino acids or comprise a sequence selected from: 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); or (b) L ₁ , L ₂ , L ₃ and L ₄ each independently comprise a sequence selected from: 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). The binding protein according to any one of claims 1 to 15, wherein L ₁ comprises the sequence GQPKAAP (SEQ ID NO: 67), L ₂ comprises the sequence TKGPS (SEQ ID NO: 68), L ₃ comprises the sequence S, and L ₄ contains the sequence RT. The binding protein according to any one of claims 1 to 15, wherein _{at least one of L 1} , L ₂ , L ₃ or L ₄ comprises the sequence DKTHT (SEQ ID NO: 66). The binding protein according to claim 19, wherein L ₁ , L ₂ , L ₃ and L ₄ comprise the sequence DKTHT (SEQ ID NO: 66). The requested item 1 to 20 binding protein according to any preceding claim, wherein said second and said third hinge -C _H2 -C _H3 polypeptide chain domains of human IgG4 hinge -C _{H2 of} -C _H3 domain, and wherein said hinge -C _H2 -C _H3 domain comprises a substitution at each amino acid at positions corresponding to positions 234 and 235 of the human IgG4 according to EU index, wherein said amino acid substitution is F234A And L235A. The requested item 1 to 20 binding protein according to any preceding claim, wherein said second and said third hinge -C _H2 -C _H3 polypeptide chain domains of human IgG4 hinge -C _{H2 of} -C _H3 domain, and wherein said hinge -C _H2 -C _H3 domains each comprise amino acid substitution at a position according to the EU index position corresponding to 233-236 of the human IgG4, wherein said amino acid substitution is E233P , F234V, L235A, and the deletion at 236. The requested item 1 to 22 according to a binding protein, wherein said second and said third hinge -C _H2 -C _H3 polypeptide chain domains of human IgG4 hinge -C _{H2 of} -C _H3 domain, and wherein said hinge -C _H2 -C _H3 domains each comprise a substituted according to the EU index at a position corresponding to the human IgG4 amino acids 228 and 409, wherein said amino acid substitution is S228P And R409K. The requested item 1 to 20 binding protein according to any preceding claim, wherein said second and said third hinge -C _H2 -C _H3 polypeptide chain domain is a human IgG1 hinge -C _{H2 of} -C _H3 domain, and wherein said hinge -C _H2 -C _H3 domain comprises a substitution at each amino acid at a position corresponding to position 234, 235 and 329 of human IgG1 based on the EU index, wherein the substituted amino acid They are L234A, L235A and P329A. The requested item 1 to 20 binding protein according to any preceding claim, wherein said second and said third hinge -C _H2 -C _H3 polypeptide chain domain is a human IgG1 hinge -C _{H2 of} -C _H3 domain, and wherein said hinge -C _H2 -C _H3 domain comprises a substitution at each amino acid at a position corresponding to position 298, 299 and 300 of human IgG1 based on the EU index, wherein the substituted amino acid It is S298N, T299A and Y300S. The requested item 1 to 25 according to any one of the binding protein, wherein the said second hinge -C _H2 -C _H3 polypeptide chain domain comprises at EU index positions 349 corresponding to the human IgG1 or IgG4 according , 366, 368, and 407, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge of the third polypeptide chain -C _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, wherein the amino acid substitutions are S354C and T366W. The requested item 1 to 25 according to any one of the binding protein, wherein the said second hinge -C _H2 -C _H3 polypeptide chain domain comprises at EU index positions 354 corresponding to the human IgG1 or IgG4 according and the amino acid substitution at position 366, wherein said amino acid substitution is a T366W S354C and; and wherein the said third hinge -C _H2 -C _H3 polypeptide chain domain comprises the corresponding index according to the EU The amino acid substitutions at positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein according to any one of claims 1 to 27. An expression vector comprising the nucleic acid molecule according to claim 28. An isolated host cell comprising the nucleic acid molecule according to claim 28 or the expression vector according to claim 29. The isolated host cell according to claim 30, wherein the host cell is a mammalian cell or an insect cell. A pharmaceutical composition comprising the binding protein according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier. A method of preventing and/or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of at least one binding protein as described in any one of claims 7 to 27 or as described in claim 32 The pharmaceutical composition. The method according to claim 33, wherein the at least one binding protein is co-administered with a chemotherapeutic agent. The method according to claim 33 or 34, wherein the patient is a human. The method according to any one of claims 33 to 35, wherein the third antigen binding site binds a human CD38 polypeptide, and wherein cancer cells from the patient express CD38. The method of claim 36, wherein the cancer is multiple myeloma. The method of claim 36, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. The method according to any one of claims 36 to 38, wherein, before administering the binding protein, the patient has been treated with daratumumab without a washout period. The method according to any one of claims 33 to 35, wherein the third antigen binding site binds to a human HER2 polypeptide, and wherein cancer cells from the patient express HER2. The method of claim 40, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC). The binding protein according to any one of claims 7 to 27 or the pharmaceutical composition according to claim 32 is used for the prevention and/or treatment of cancer in a patient. The binding protein for the use or the composition for the use according to claim 42, wherein the at least one binding protein is co-administered with a chemotherapeutic agent. The binding protein for the use or the composition for the use according to claim 42 or claim 43, wherein the patient is a human. The binding protein for the use or the composition for the use according to any one of claims 42 to 44, wherein the third antigen binding site binds to a human CD38 polypeptide, and wherein The patient's cancer cells showed CD38. The binding protein for the use or the composition for the use according to claim 45, wherein the cancer is multiple myeloma. The binding protein for the use or the composition for the use according to claim 45, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia Globular leukemia (CLL) or B-cell lymphoma. The binding protein for the use or the composition for the use according to any one of claims 45 to 47, wherein, before the administration of the binding protein, the patient is already in the absence of clearance period In the case of treatment with daralimumab. The binding protein for the use or the composition for the use according to any one of claims 42 to 44, wherein the third antigen binding site binds to a human HER2 polypeptide, and wherein The patient's cancer cells showed HER2. The binding protein for the use or the composition for the use according to claim 49, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC). A method for expanding T cells, the method comprising contacting T cells with a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises The structure of V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain contains the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _H1 -hinge -C _H2 -C _H3 [II] and a third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _H1 - hinge -C _H2 -C _H3 [III] and a fourth polypeptide chain comprises represented by the formula The structure of: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the variable domain of the second immunoglobulin light chain; V _L3 is the third immunoglobulin Protein 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 an immunoglobulin light chain constant domain; C C _{Hl Hl} immunoglobulin heavy chain constant domain; C C _{H2 of H2 of} immunoglobulin heavy chain constant domain; C _{H3 of} an immunoglobulin C _H3 heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; wherein said formula I The polypeptide and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen-binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 form the second that binds to the CD3 polypeptide An antigen binding site, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and a CDR containing the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56) -H2 sequence containing RGVYYALSPFDY (SEQ ID NO: 57) CDR-H3 sequence of the amino acid sequence and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ is a or L and X ₃ is the CDR-L1 sequence of the amino acid sequence of Q, R or F QSLVHX ₁ NX ₂ X ₃ TY (SEQ ID NO: 180), the CDR-L1 sequence containing the amino acid sequence of KVS (SEQ ID NO: 64) The L2 sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding site that binds to the CD38 polypeptide. The method according to claim 51, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding the CAR. The method according to claim 51 or claim 52, wherein the T cell is a memory T cell or an effector T cell. A method for amplifying virus-specific memory T cells, the method comprising contacting virus-specific memory T cells with a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first The polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and fourth The polypeptide chain comprises the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the variable domain of the second immunoglobulin light chain ; 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 first immunoglobulin heavy chain variable domain; three immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain ; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ are amino acids Linker; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 forms a second antigen-binding site that binds to the CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), and a CDR-H1 sequence containing IKDKSNSYAT (SEQ ID NO: 56). ) a CDR-H2 sequence comprising the amino acid sequence and RGVYYALSPFDY (SEQ ID NO: 57) sequences of CDR-H3 amino acid sequences, and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ is A or L and X ₃ is Q, R or F QSLVHX ₁ NX ₂ X ₃ CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), containing KVS (SEQ ID NO: 64) The amino acid sequence of CDR-L2 sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding site that binds to the CD38 polypeptide. The method according to claim 54, wherein the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo. The method according to claim 54 or claim 55, wherein the contact line of the virus-specific memory T cell with the binding protein causes activation and/or proliferation of the virus-specific memory T cell. A method for treating chronic viral infections, the method comprising administering to a patient in need an effective amount of a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain Contains the structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -Hinge} -C _H2 -C _H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _{H1 -Hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain Contains the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the variable domain of the second immunoglobulin light chain; 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 protein heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; Wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 form a binding The second antigen-binding site of a CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), an amine containing IKDKSNSYAT (SEQ ID NO: 56) CDR-H2 sequence comprising amino acid sequence and RGVYYALSPFDY (SEQ ID NO: 57) CDR-H3 amino acid sequences of the sequences, and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ is _{The CDR-L1 sequence of the amino acid sequence of QSLVHX 1} NX ₂ X ₃ TY (SEQ ID NO: 180) with A or L and X ₃ being Q, R or F, and the amino group containing KVS (SEQ ID NO: 64) The CDR-L2 sequence of the acid sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding site that binds to the CD38 polypeptide. The method of claim 57, wherein the patient is a human. The method according to claim 57 or claim 58, wherein the binding protein is administered to the patient in a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier. The method of any one of claims 57 to 59, wherein the administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the patient. The method according to any one of claims 53 to 56 and 60, wherein the memory T cell is a CD8+ or CD4+ memory T cell. The method according to any one of claims 53 to 56, 60, and 61, wherein the memory T cell is a central memory T cell (T _CM ) or an effector memory T cell (T _EM ). The method according to any one of claims 54 to 62, wherein the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papilloma virus (HPV), Estane-Barr virus (EBV), human foam virus (HFV), herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2). The method of any one of claims 51 to 63, wherein the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide. A binding protein for use in the expansion of T cells, wherein the binding protein is to be contacted with T cells, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain Contains the structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -Hinge} -C _H2 -C _H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _{H1 -Hinge} -C _H2 -C _H3 [III] and the fourth polypeptide chain Contains the structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the variable domain of the second immunoglobulin light chain; 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 protein heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ is a linker amino acid; Wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 form a binding The second antigen-binding site of a CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), an amine containing IKDKSNSYAT (SEQ ID NO: 56) CDR-H2 sequence comprising amino acid sequence and RGVYYALSPFDY (SEQ ID NO: 57) CDR-H3 amino acid sequences of the sequences, and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ is _{The CDR-L1 sequence of the amino acid sequence of QSLVHX 1} NX ₂ X ₃ TY (SEQ ID NO: 180) with A or L and X ₃ being Q, R or F, and the amino group containing KVS (SEQ ID NO: 64) The CDR-L2 sequence of the acid sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding site that binds to the CD38 polypeptide. The binding protein for the use according to claim 65, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or contains a polynucleotide encoding the CAR. The binding protein for the use according to claim 65 or claim 66, wherein the T cell is a memory T cell or an effector T cell. A binding protein used in the amplification of virus-specific memory T cells, wherein the binding protein is to be brought into contact with virus-specific memory T cells, wherein the binding protein includes four polypeptide chains forming three antigen binding sites, Wherein the first polypeptide chain comprises a structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain comprises a structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] And the fourth polypeptide chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; 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 a third immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ are An amino acid linker; wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 to form a second antigen binding site binds to CD3 polypeptide, wherein said domain comprises V _H2: comprising GFTFTKAW (SEQ ID NO: 55) the amino acid sequence of a CDR-H1 sequence comprising IKDKSNSYAT (SEQ ID NO: CDR-H2 sequence 56) and comprising the amino acid sequence RGVYYALSPFDY (SEQ ID NO: CDR- H3 sequence 57) the amino acid sequence and the V _L2 domain comprises: wherein X ₁ is E comprising Or Q, X ₂ is A or L and X ₃ is Q, R or F QSLVHX ₁ NX ₂ X ₃ CDR-L1 sequence of the amino acid sequence of TY (SEQ ID NO: 180), containing KVS (SEQ ID NO : 64) the CDR-L2 sequence of the amino acid sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding to the CD38 polypeptide Site. The binding protein for the use according to claim 68, wherein the virus-specific memory T cell is to be contacted with the binding protein in vitro or ex vivo. The binding protein for the use according to claim 68 or claim 69, wherein the contact system of the virus-specific memory T cell and the trispecific binding protein causes the activation of the virus-specific memory T cell and / Or proliferation. A binding protein used in the treatment of chronic viral infections, wherein the binding protein is to be administered to patients in need, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide The chain contains the structure represented by the following formula: V _L2 -L ₁ -V _L1 -L ₂ -C _L [I] and the second polypeptide chain contains the structure represented by the following formula: V _H1 -L ₃ -V _H2 -L ₄ -C _{H1 -hinge} -C _H2 -C _H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V _H3 -C _{H1 -hinge} -C _H2 -C _H3 [III] and the fourth polypeptide The chain comprises a structure represented by the following formula: V _L3 -C _L [IV] where: V _L1 is the variable domain of the first immunoglobulin light chain; V _L2 is the variable domain of the second immunoglobulin light chain; 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 immunoglobulin heavy chain variable domain; C _L is an immunoglobulin light chain constant domain; C _H1 C _H1 is an immunoglobulin heavy chain constant domain; C _H2 C _H2 is an immunoglobulin heavy chain constant domain; C _H3 C _H3 is an immunoglobulin heavy chain constant domain; a hinge connecting the C _H1 is an immunoglobulin hinge region domain, C _H2; and L _1, L _2, L ₃ and L ₄ is a linker amino acid ; Wherein the polypeptide of formula I and the polypeptide of formula II form a staggered light chain-heavy chain pair; and wherein V _H1 and V _L1 form the first antigen binding site that binds to the CD28 polypeptide; wherein V _H2 and V _L2 form The second antigen-binding site that binds to the CD3 polypeptide, wherein the V _H2 domain comprises: a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H1 sequence containing IKDKSNSYAT (SEQ ID NO: 56) CDR-H2 sequence comprising the amino acid sequence and RGVYYALSPFDY (SEQ ID NO: 57) CDR-H3 amino acid sequences of the sequences, and the V _L2 domain comprises: comprising wherein X ₁ is E or Q, X ₂ The CDR-L1 sequence of the amino acid sequence of _{QSLVHX 1} NX ₂ X ₃ TY (SEQ ID NO: 180), which is A or L and X ₃ is Q, R or F, and an amine containing KVS (SEQ ID NO: 64) The CDR-L2 sequence of the base acid sequence and the CDR-L3 sequence containing the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and wherein V _H3 and V _L3 form a third antigen binding site that binds to the CD38 polypeptide. The binding protein for the use according to claim 71, wherein the patient is a human. The binding protein for the use according to claim 71 or claim 72, wherein the binding protein is to be administered to the binding protein as a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier patient. The binding protein for the use according to any one of claims 71 to 73, wherein administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the patient. The binding protein for the use according to any one of claims 67 to 70 and 74, wherein the memory T cell is a CD8+ or CD4+ memory T cell. The binding protein for the use according to any one of claims 67 to 70, 74 and 75, wherein the memory T cell is a central memory T cell (T _CM ) or an effector memory T cell (T _EM ) . The binding protein for the use according to any one of claims 68 to 76, wherein the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus ( HBV), human papilloma virus (HPV), estan-Barr virus (EBV), human foam virus (HFV), herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2). The binding protein for the use according to any one of claims 65 to 77, wherein the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide. The method or binding protein for the use according to any one of claims 51 to 78, wherein the V _H1 domain comprises: CDR- containing the amino acid sequence of GYTFTSYY (SEQ ID NO: 49) H1 sequence, CDR-H2 sequence containing the amino acid sequence of IYPGNVNT (SEQ ID NO: 50) and CDR-H3 sequence containing the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51); and the V _L1 domain Containing: the CDR-L1 sequence containing the amino acid sequence of QNIYVW (SEQ ID NO: 52), the CDR-L2 sequence containing the amino acid sequence of KAS (SEQ ID NO: 53) and the QQGQTYPY sequence containing QQGQTYPY (SEQ ID NO: 54 ) CDR-L3 sequence of the amino acid sequence. The request for the use of the method of item 79, or a binding protein, wherein said domain comprising V _H1 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91) The amino acid sequence and / or the V _L1 domain comprises The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92). The requested item 51 to 80 The method of any one of for use or binding proteins, wherein the sequence of the CDR-L1 V _L2 domain comprising the amino acid sequence selected from the following: QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61) and QSLVHENLRTY (SEQ ID NO: 62). The method for the use as a binding protein or the requested item 81, wherein said V _H2 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302) amino acid sequence, and / V _L2 or said domain comprises the amino acid sequence selected from the following: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95 ), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO : 98). The method or binding protein for the use according to any one of claims 51 to 82, wherein: (a) the V _H3 domain comprises: an amino acid containing GYTFTSYA (SEQ ID NO: 13) The CDR-H1 sequence of the sequence, the CDR-H2 sequence containing the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and the CDR-H3 sequence containing the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and The V _L3 domain includes: the CDR-L1 sequence containing the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), the CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 17) and the QQNKEDPWT (SEQ ID NO: 17) ID NO: 18) CDR-L3 sequence of the amino acid sequence; (b) The V _H3 domain contains: the CDR-H1 sequence containing the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), and contains FDPEDGET ( SEQ ID NO: CDR-H2 sequence 20) and comprising the amino acid sequence TTGRFFDWF (SEQ ID NO: CDR- H3 sequence 21) of the amino acid sequence; V _L3 and said domain comprises: comprising QSVISRF (SEQ ID NO: 22) CDR-L1 sequence of the amino acid sequence, CDR-L2 sequence containing the amino acid sequence of GAS (SEQ ID NO: 23) and amino acid sequence containing QQDSNLPIT (SEQ ID NO: 24) (C) The V _H3 domain contains: the CDR-H1 sequence containing the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), the amino acid containing INPGSGST (SEQ ID NO: 26) CDR-H2 sequence comprising the sequence and ARYAYGY (SEQ ID NO: 27) CDR-H3 amino acid sequences of the sequence; V _L3 and said domain comprises: comprising QNVGTA (SEQ ID NO: 28) the amino acid sequence of The CDR-L1 sequence, the CDR-L2 sequence containing the amino acid sequence of SAS (SEQ ID NO: 29) and the CDR-L3 sequence containing the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30); (d) The V _H3 domain includes: the CDR-H1 sequence containing the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), the CDR-H2 sequence containing the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and the CDR-H2 sequence containing the amino acid sequence of ARRFEGFYYSMDY ( SEQ ID NO: 33) CDR-H3 sequence of the amino acid sequence Column; V _L3 and said domain comprises: comprising QSLVHSNGNTY (SEQ ID NO: 34) amino acid sequences of CDR-L1 sequence, comprising KVS (SEQ ID NO: 35) CDR-L2 of the sequence of the amino acid sequence And a CDR-L3 sequence containing the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36); (e) the V _H3 domain contains: CDR-H1 containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 37) Sequence, the CDR-H2 sequence containing the amino acid sequence of IWYDGSNK (SEQ ID NO: 38) and the CDR-H3 sequence containing the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and the V _L3 domain comprises : CDR-L1 sequence containing the amino acid sequence of QGISSY (SEQ ID NO: 40), CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 41) and QGLNSFPYT (SEQ ID NO: 42) The CDR-L3 sequence of the amino acid sequence; or (f) the V _H3 domain comprises: the CDR-H1 sequence containing the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), and the CDR-H1 sequence containing IWYDGSNK (SEQ ID NO: CDR-H2 sequence 44) and comprising the amino acid sequence ARMFRGAFDY (SEQ ID NO: CDR- H3 sequence 45) the amino acid sequence; V _L3 and said domain comprises: comprising QGIRND (SEQ ID NO: 46 ) The CDR-L1 sequence of the amino acid sequence, the CDR-L2 sequence containing the amino acid sequence of AAS (SEQ ID NO: 47) and the CDR-L3 containing the amino acid sequence of LQDYIYYPT (SEQ ID NO: 48) sequence. The method or the binding protein for the use according to claim 83, wherein: (a) the V _H3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGTL or VSS (SEQ ID NO: RAY and VSS) of the SEQ ID NO: RAY and VSS VT: LRFTY: 79 _L3 domain comprises DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80) amino acid sequences; (b) the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81) amino acid sequence and / or structure of the V _L3 domain comprises EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82) the amino acid sequence; (c) the V _H3 domain comprises QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83) amino acid sequences, and / or the V _L3 domain comprises The amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84) (SEQ ID NO: 84); (d) the V _H3 domain contains QVQLVQYYGYYGGDHMGVRMGVRKAWVISPNYGDAVSGV YAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85) The amino acid sequence and / or the V _L3 domain comprises DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86) amino acid sequences; (e) the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS ( SEQ ID NO: 87) the amino acid sequence and / or the V _L3 domain comprises DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88) the amino acid sequence; or (f) the V _H3 domain comprises QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89) the amino acid sequence and / or the V _L3 domain comprises AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90) the amino acid sequence. The method or binding protein for the use according to any one of claims 51 to 82, wherein: (a) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid of SEQ ID NO: 158 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 159 or the amino acid sequence of SEQ ID NO: 159 The amino acid sequence is at least 95% identical to the amino acid sequence; (b) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid of SEQ ID NO: 162 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 163 or the amino acid sequence of SEQ ID NO: 163 The amino acid sequence is at least 95% identical to the amino acid sequence; (c) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid of SEQ ID NO: 166 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 167 or the amino acid sequence of SEQ ID NO: 167 The amino acid sequence is at least 95% identical to the amino acid sequence; (d) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid of SEQ ID NO: 170 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 171 or the amino acid sequence of SEQ ID NO: 171 The amino acid sequence is at least 95% identical to the amino acid sequence; (e) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid of SEQ ID NO: 174 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 175 or the amino acid sequence of SEQ ID NO: 175 The amino acid sequence is at least 95% identical to the amino acid sequence; (f) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid of SEQ ID NO: 178 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 179 or the amino acid sequence of SEQ ID NO: 179 The amino acid sequence is at least 95% identical to the amino acid sequence; (g) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid of SEQ ID NO: 183 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 184 or the amino acid sequence of SEQ ID NO: 184 The amino acid sequence is at least 95% identical to the amino acid sequence; or (h) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain It comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid of SEQ ID NO: 187 Sequence or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 188 or the amino acid sequence of SEQ ID NO: 188 The amino acid sequence is at least 95% identical to the amino acid sequence. A vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein according to any one of claims 1-27. The vector system according to claim 86, wherein the vector system includes a first vector encoding the first polypeptide chain of the binding protein, and a second vector encoding the second polypeptide chain of the binding protein. A vector, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. A set of polynucleotides, the set of polynucleotides comprising: (a) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 189, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 190, the polynucleotide containing SEQ ID NO: 191 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 192; (b) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 193, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 194, the polynucleotide containing SEQ ID NO: 195 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 196; (c) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 197, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 198, the polynucleotide containing SEQ ID NO: 199 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 200; (d) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 201, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 202, and the polynucleotide containing SEQ ID NO: 203 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 204; (e) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 205, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 206, the polynucleotide containing SEQ ID NO: 207 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 208; (f) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 209, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 210, the polynucleotide containing SEQ ID NO: 211 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 212; (g) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 213, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 214, the polynucleotide containing SEQ ID NO: 215 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 216; (h) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 217, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 218, the polynucleotide containing SEQ ID NO: 219 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 220; (i) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 221, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 222, the polynucleotide containing SEQ ID NO: 223 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 224; (j) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 225, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 226, the polynucleotide containing SEQ ID NO: 227 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 228; (k) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 229, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 230, the polynucleotide containing SEQ ID NO: 231 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 232; (1) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 233, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 234, the polynucleotide containing SEQ ID NO: 235 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 236; (m) the first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 237, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 238, the polynucleotide containing SEQ ID NO: 239 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 240; (n) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 241, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 242, the polynucleotide containing SEQ ID NO: 243 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 244; (o) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 245, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 246, the polynucleotide containing SEQ ID NO: 247 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 248; (p) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 249, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 250, the polynucleotide containing SEQ ID NO: 251 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 252; (q) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 253, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 254, the polynucleotide containing SEQ ID NO: 255 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 256; (r) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 257, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 258, the polynucleotide containing SEQ ID NO: 259 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 260; (s) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 261, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 262, the polynucleotide containing SEQ ID NO: 263 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 264; (t) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 265, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 266, the polynucleotide containing SEQ ID NO: 267 The third polynucleotide of the acid sequence and the 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 second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 270, the polynucleotide containing SEQ ID NO: 271 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 272; or (v) The first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 273, the second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 274, the polynucleotide containing SEQ ID NO: 275 The third polynucleotide of the acid sequence and the fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 276. The kit according to claim 88, wherein the first, second, third and fourth polynucleotides are present on one or more expression vectors.

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