Anti-CD28 antibody and use thereof

Abstract

Provided are an anti-CD28 antibody or an antigen-binding fragment thereof, and a multi-specific antibody constructed on the basis of the antibody or the antigen-binding fragment thereof. The multi-specific antibody comprises an anti-CD3×CD28 bispecific antibody and an anti-tumor-associated antigen (TAA)×CD3×CD28 trispecific antibody. Further provided are a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector and a cell comprising the nucleic acid molecule, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and the use of the antibody or the antigen-binding fragment thereof, in particular for the treatment of tumors or immunodeficiency diseases.

Description

Anti-CD28 antibodies and uses thereof

[0001] This application claims priority to Chinese patent application 202410544396X with the filing date of 2024 / 4 / 30. This application incorporates the entire text of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the field of antibody drugs in the field of biological medicine. Specifically, the present application relates to antibodies capable of specifically binding to human CD28, including multispecific antibodies. BACKGROUND

[0003] T cells play an important role in the process of the immune system resisting the invasion of foreign pathogens and eliminating abnormal cells in the body, therefore, activating T cells is one of the difficult problems that need to be solved in many immune type drugs. In many examples, co-stimulatory receptors or their ligands are introduced into immunotherapy as a "second signal" of T cell activation to enhance the activation level of TCR-CD3 on T cells.

[0004] CD28 is a co-stimulatory receptor expressed on the surface of T cells, which induces the differentiation, proliferation and secretion of IL-2 and other cytokines of T cells by mediating co-stimulatory signals, and is of great significance to the design and application of immunotherapy drugs. However, in the process of immunotherapy, CD28 needs an activation signal, and cannot be completely applied to the design scheme of traditional inhibitory or blocking antibodies, therefore, the screening of agonist type anti-CD28 antibodies is more difficult than that of traditional antibodies. On the other hand, whether anti-CD28 antibodies are used as T cell activation antibodies in combination with other drugs, or anti-CD28 antibody fragments are used as a domain in T cell engager type multispecific antibodies, it is hoped to find effective and highly versatile anti-CD28 antibodies. It can be seen that there is still an unmet need in the art for agonist type anti-CD28 antibodies with ideal effects.

[0005] T cell engager (TCE) antibodies against tumor-associated antigens (TAAs) are a new and promising immunotherapy. Its main mechanism of action is to tightly connect cytotoxic T cells with tumor cells through TCE antibodies, thereby forming an artificial immune contact, redirecting the cytotoxic activity of T cells to tumor cells, and ultimately leading to selective attack and lysis of targeted tumor cells. In the prior art, CD3 + TCE is the most common form of T cell engager antibodies, CD3 +TCEs have achieved certain results in the immunotherapy of tumors, but there are also some problems, such as (1) exhaustion or apoptosis of T cells after activation, poor persistence of efficacy; (2) low specificity of TAA selection, and it is a challenge for TCEs to select an antigen target that recognizes tumor cell specific antigens but does not recognize normal tissue cells; (3) the production of anti-drug antibodies caused by the immunogenicity of antibody drugs is one of the main reasons for the failure of TCEs drugs in clinical use. It can be seen that there is still an unmet need in the art for more ideal anti-tumor effects of TCE antibodies with low immunogenicity. SUMMARY

[0006] In order to more effectively improve the activation of antibodies to T cells and mediate more effective killing of tumor cells, the present application provides a new antibody or antigen binding fragment thereof specifically binding to CD28. The inventors of the present application immunize test animals with human CD28 (huCD28) antigens, isolate B lymphocytes producing CD28-activated antibodies, and obtain a new antibody or antigen binding fragment thereof specifically binding to CD28 accordingly. Through binding property tests and their application in bispecific antibodies or TAAxCD3xCD28 trispecific antibodies, it is found that the antibody or antigen binding fragment thereof specifically binding to CD28 screened in the present application has (1) high affinity to CD28 target antigen; (2) promotes the binding of TAA binding domain to its target antigen; (3) stronger ability to induce T cell activation and T cell proliferation and differentiation; (4) mediates more effective tumor killing and in vivo tumor inhibition, thereby completing the present application.

[0007] Meanwhile, the inventors of the present application also obtain new, humanized anti-MSLN antibodies and humanized anti-PSMA antibodies based on the variable region sequences of anti-MSLN mouse antibodies and anti-PSMA mouse antibodies, respectively, through humanization modification, providing a selection of low immunogenicity antibodies for the treatment of corresponding diseases. These two antibodies against TAA can not only be used as a domain in TCEs for targeting tumors, but also be used as monospecific antibodies.

[0008] In one aspect, the present application provides an antibody or antigen binding fragment thereof specifically binding to CD28, comprising a domain specifically binding to CD28.

[0009] In a preferred embodiment, the domain specifically binding to CD28 comprises:

[0010] (1) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) as shown in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) as shown in SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (HCDR3) as shown in SEQ ID NO: 13; and

[0011] (2) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 19, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 25, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 31.

[0012] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 37 or SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 37 or SEQ ID NO: 38.

[0013] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 48 or SEQ ID NO: 49, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 48 or SEQ ID NO: 49.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the following: a Fab, a Fv, a F(ab’)2, a single-chain Fv (scFv), a monospecific antibody, a multispecific antibody, a human antibody, a humanized antibody, and a chimeric antibody.

[0015] For example, the antibody or antigen-binding fragment thereof is a scFv that specifically binds to CD28.

[0016] In some embodiments, the scFv that specifically binds to CD28 comprises the VH and the VL connected by an intradomain linker. In some embodiments, the intradomain linker is selected from a first linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, the intradomain linker is SEQ ID NO: 213.

[0017] In some embodiments, the CD28 is of mammalian origin. Preferably, the CD28 is of primate origin. For example, the CD28 is of human origin. Specifically, the antibody or antigen-binding fragment thereof specifically binds to human CD28.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof is a monospecific antibody. Specifically, it is an anti-CD28 antibody. The anti-CD28 antibody, based on the property of specifically binding to CD28, has the function of promoting T cell activation, which is of great significance in the treatment of tumors and immunodeficiency diseases.

[0019] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds CD28 and a tumor-associated antigen (TAA). Specifically, the TAA is selected from the group consisting of EGFR, Her2, Her3, Her4, NY-ESO-1, B7-H3, CD19, CD20, CD22, CD30, CD33, CD46, CD70, CEACAM5, ENPP3, PSMA (FOLH1), c-Met, folate receptor alpha, GPC3, Claudin 18.2, MUC1, MUC2, MUC4, MUC5AC, MUC6, MUC16, MUC20, NCAM, Nectin4, 5T4, IL13ra2, LIV1 (ZIP6), CD123, CD7, GD2, PSCA, P-cadherin, STEAP-1, SLC44A4, SLITRK6, TROP2, EBV16-E7, H3.3, EGFRvIII, BCMA, and MSLN. Preferably, the TAA is selected from PSMA, MSLN, 5T4, and EGFR.

[0020] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds CD28 and another T cell surface molecule.

[0021] In certain embodiments, the other T cell surface molecule is a molecule expressed on the membrane surface of a T cell other than CD28. For example, the T cell surface molecule is selected from the group consisting of CD2, CD3, CD4, CD8, CD40, CD137, OX40, and GITR. For another example, the T cell surface molecule is CD3. Specifically, the antibody or antigen-binding fragment thereof comprises a domain that specifically binds CD28 and a domain that specifically binds CD3.

[0022] In preferred embodiments, the domain that specifically binds CD3 comprises:

[0023] (1) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and

[0024] (2) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3).

[0025] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 205, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 205.

[0026] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 206, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 206.

[0027] In some embodiments, the domain that specifically binds to CD28 and the domain that specifically binds to CD3 are a scFv that specifically binds to CD28 and a scFv that specifically binds to CD3, respectively.

[0028] In some embodiments, the scFv that specifically binds to CD3 comprises the VH and the VL connected by an intradomain linker. In some embodiments, the scFv that specifically binds to CD28 comprises the VH and the VL connected by an intradomain linker. In some embodiments, the intradomain linker is selected from a first linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 213.

[0029] In some embodiments, the scFv that specifically binds to CD3 is connected to the scFv that specifically binds to CD28 by an interdomain linker. In some embodiments, the interdomain linker is selected from a second linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219. Preferably, SEQ ID NO: 217.

[0030] In some embodiments, the scFv that specifically binds to CD3 is located at the N-terminus of the scFv that specifically binds to CD28.

[0031] In some embodiments, the scFv that specifically binds to CD3 is located at the C-terminus of the scFv that specifically binds to CD28.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. Specifically, the bispecific antibody is an anti-CD3xCD28 antibody.

[0033] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to TAA, CD3 and CD28. It comprises:

[0034] (1) a first binding domain capable of specifically binding to a tumor associated antigen (TAA), the first binding domain comprising a heavy chain variable region (VH TAA ) and a light chain variable region (VL TAA );

[0035] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0036] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0037] In certain embodiments, the TAA is selected from one or more of EGFR, Her2, Her3, Her4, NY-ESO-1, B7-H3, CD19, CD20, CD22, CD30, CD33, CD46, CD70, CEACAM5, ENPP3, PSMA (FOLH1), c-Met, folate receptor alpha, GPC3, Claudin 18.2, MUC1, MUC2, MUC4, MUC5AC, MUC6, MUC16, MUC20, NCAM, Nectin4, 5T4, IL13ra2, LIV1 (ZIP6), CD123, CD7, GD2, PSCA, P-cadherin, STEAP-1, SLC44A4, SLITRK6, TROP2, EBV16-E7, H3.3, EGFRvIII, BCMA and MSLN. Preferably, the TAA is selected from PSMA, MSLN, EGFR and 5T4.

[0038] In the selection of specific TAAs, the present application intends to cover as many tumor types as possible to demonstrate the universality of the anti-TAAxCD3xCD28 antibody protected by the present application in the selection of TAAs, and therefore, targets expressed in tumors in different parts are selected. Specifically, PSMA is overexpressed in prostate tumors; MSLN is overexpressed in mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma and cholangiocarcinoma, etc.; EGFR is overexpressed in gastric cancer, pancreatic cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer, head and neck squamous cell carcinoma and glioma, etc.; 5T4 is overexpressed in non-small cell lung cancer, renal cancer and pancreatic cancer, etc.

[0039] In some embodiments, the first binding domain is a scFv that specifically binds to a TAA.

[0040] In some embodiments, the second binding domain is a scFv that specifically binds to CD3.

[0041] In some embodiments, the third binding domain is a scFv that specifically binds to CD28.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. Specifically, the trispecific antibody is an anti-TAAxCD3xCD28 antibody.

[0043] The trispecific antibody provided herein has at least the following advantages: (1) the trispecific antibody is a complex structure capable of binding multiple targets, co-activating CD3 + T-cell Engagers (TCEs) have been proven to have ideal target-specific binding ability, T-cell activation, T-cell proliferation promotion, target cell killing activity, and other positive effects on various solid tumor cells, especially enhanced tumor infiltration; (2) the antibody or antigen-binding fragment thereof has excellent effectiveness while successfully solving the problem of T-cell death after single signal activation in the field of treating solid tumors, and can provide more persistent therapeutic effect, which has significant meaning for clinical application.

[0044] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to PSMA, CD3 and CD28. It comprises:

[0045] (1) a first binding domain capable of specifically binding to PSMA, the first binding domain comprising a heavy chain variable region (VH PSMA ) and a light chain variable region (VL PSMA );

[0046] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0047] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0048] In some embodiments, the first binding domain comprises:

[0049] (1) a heavy chain variable region (VHPSMA ), comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 4, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 10, SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 16; and

[0050] (2) a light chain variable region (VL PSMA ), comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 22 or SEQ ID NO: 224, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 28, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 34.

[0051] In certain embodiments, the VH PSMA comprises an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241, or an amino acid sequence that has at least 90% sequence homology to an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241.

[0052] In certain embodiments, the VL PSMA comprises an amino acid sequence as set forth in any one of SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO: 243, or an amino acid sequence that has at least 90% sequence homology to an amino acid sequence as set forth in any one of SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO: 243.

[0053] In certain embodiments, the first binding domain is a scFv that specifically binds PSMA.

[0054] In certain embodiments, the second binding domain is a scFv that specifically binds CD3.

[0055] In certain embodiments, the third binding domain is a scFv that specifically binds CD28.

[0056] In certain embodiments, the first binding domain comprises a VH PSMA and a VL PSMA connected by an intra-domain linker.

[0057] In certain embodiments, the second binding domain comprises a VH CD3 and a VLCD3 .

[0058] In certain embodiments, the third binding domain comprises a VH CD28 and a VL CD28 .

[0059] In certain embodiments, the intradomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 213.

[0060] In certain embodiments, the first binding domain is connected to the second binding domain by a first interdomain linker.

[0061] In certain embodiments, the first interdomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 216.

[0062] In certain embodiments, the second binding domain is connected to the third binding domain by a second interdomain linker.

[0063] In certain embodiments, the second interdomain linker is selected from a second linker sequence. The second linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219. Preferably, SEQ ID NO: 217.

[0064] In certain embodiments, the first binding domain is located N-terminal to the second binding domain.

[0065] In certain embodiments, the second binding domain is located N-terminal to the third binding domain.

[0066] In certain embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. Specifically, the trispecific antibody is an anti-PSMAxCD3xCD28 antibody.

[0067] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to MSLN, CD3 and CD28. It comprises:

[0068] (1) a first binding domain capable of specifically binding to MSLN, the first binding domain comprising a heavy chain variable region (VH MSLN ) and a light chain variable region (VL MSLN );

[0069] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0070] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0071] In certain embodiments, the first binding domain comprises:

[0072] (1) a heavy chain variable region (VH MSLN ) comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 3, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 9, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 15; and

[0073] (2) a light chain variable region (VL MSLN ) comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 21, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 27, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 33.

[0074] In certain embodiments, the VH MSLN comprises an amino acid sequence as set forth in SEQ ID NO: 45, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43, or an amino acid sequence that comprises at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 45, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

[0075] In certain embodiments, the VL MSLNcomprises an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 55, or an amino acid sequence that includes at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 55.

[0076] In certain embodiments, the first binding domain is an scFv that specifically binds MSLN.

[0077] In certain embodiments, the second binding domain is an scFv that specifically binds CD3.

[0078] In certain embodiments, the third binding domain is an scFv that specifically binds CD28.

[0079] In certain embodiments, the first binding domain comprises a VH MSLN and a VL MSLN connected by an intradomain linker.

[0080] In certain embodiments, the second binding domain comprises a VH CD3 and a VL CD3 connected by an intradomain linker.

[0081] In certain embodiments, the third binding domain comprises a VH CD28 and a VL CD28 connected by an intradomain linker.

[0082] In certain embodiments, the intradomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 213.

[0083] In certain embodiments, the first binding domain is connected to the second binding domain by a first interdomain linker.

[0084] In certain embodiments, the first interdomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 216.

[0085] In certain embodiments, the second binding domain is connected to the third binding domain by a second interdomain linker.

[0086] In certain embodiments, the second interdomain linker is selected from a second linker sequence. The second linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219. Preferably, SEQ ID NO: 217.

[0087] In certain embodiments, the first binding domain is located N-terminal to the second binding domain.

[0088] In certain embodiments, the second binding domain is located N-terminal to the third binding domain.

[0089] In certain embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. Specifically, the trispecific antibody is an anti-MSLNxCD3xCD28 antibody.

[0090] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to 5T4, CD3 and CD28. It comprises:

[0091] (1) a first binding domain capable of specifically binding to 5T4, the first binding domain comprising a heavy chain variable region (VH 5T4 ) and a light chain variable region (VL 5T4 );

[0092] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0093] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0094] In certain embodiments, the first binding domain comprises:

[0095] (1) a heavy chain variable region (VH 5T4 ) comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 6, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 12, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 18; and

[0096] (2) a light chain variable region (VL 5T4 comprises a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 24, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 30, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 36.

[0097] In certain embodiments, the VH 5T4 comprises an amino acid sequence as set forth in SEQ ID NO: 47, or an amino acid sequence that comprises at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 47.

[0098] In certain embodiments, the VL 5T4 comprises an amino acid sequence as set forth in SEQ ID NO: 57, or an amino acid sequence that comprises at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 57.

[0099] In certain embodiments, the first binding domain is a scFv that specifically binds 5T4.

[0100] In certain embodiments, the second binding domain is a scFv that specifically binds CD3.

[0101] In certain embodiments, the third binding domain is a scFv that specifically binds CD28.

[0102] In certain embodiments, the first binding domain comprises a VH 5T4 and a VL 5T4 connected by an intradomain linker.

[0103] In certain embodiments, the second binding domain comprises a VH CD3 and a VL CD3 connected by an intradomain linker.

[0104] In certain embodiments, the third binding domain comprises a VH CD28 and a VL CD28 connected by an intradomain linker.

[0105] In certain embodiments, the intradomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 213.

[0106] In certain embodiments, the first binding domain is connected to the second binding domain by a first interdomain linker.

[0107] In certain embodiments, the first interdomain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 216.

[0108] In certain embodiments, the second binding domain is connected to the third binding domain by a second interdomain linker.

[0109] In certain embodiments, the second interdomain linker is selected from a second linker sequence. The second linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219. Preferably, SEQ ID NO: 217.

[0110] In certain embodiments, the first binding domain is located N-terminal to the second binding domain.

[0111] In certain embodiments, the second binding domain is located N-terminal to the third binding domain.

[0112] In certain embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. Specifically, the trispecific antibody is an anti-5T4xCD3xCD28 antibody.

[0113] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to EGFR, CD3 and CD28. It comprises:

[0114] (1) a first binding domain capable of specifically binding to EGFR, the first binding domain comprising a heavy chain variable region (VH EGFR ) and a light chain variable region (VL EGFR );

[0115] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0116] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0117] In certain embodiments, the first binding domain comprises:

[0118] (1) a heavy chain variable region (VH EGFR ) comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 5, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 11, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 17; and

[0119] (2) a light chain variable region (VL EGFR ) comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 23, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 29, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 35.

[0120] In certain embodiments, the VH EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 46, or an amino acid sequence that comprises at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 46.

[0121] In certain embodiments, the VL EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 56, or an amino acid sequence that comprises at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 56.

[0122] In certain embodiments, the first binding domain is a scFv that specifically binds EGFR.

[0123] In certain embodiments, the second binding domain is a scFv that specifically binds CD3.

[0124] In certain embodiments, the third binding domain is a scFv that specifically binds CD28.

[0125] In certain embodiments, the first binding domain comprises a VH EGFR and a VL EGFR connected by an intra-domain linker.

[0126] In certain embodiments, the second binding domain comprises a VH CD3 and a VL CD3 connected by an intra-domain linker.

[0127] In certain embodiments, the third binding domain comprises a VH CD28and VL CD28 .

[0128] In certain embodiments, the intra-domain linker within the first binding domain is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 213.

[0129] In certain embodiments, the first binding domain is connected to the second binding domain by a first inter-domain linker.

[0130] In certain embodiments, the first inter-domain linker is selected from a first linker sequence. The first linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216. Preferably, SEQ ID NO: 216.

[0131] In certain embodiments, the second binding domain is connected to the third binding domain by a second inter-domain linker.

[0132] In certain embodiments, the second inter-domain linker is selected from a second linker sequence. The second linker sequence comprises any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219. Preferably, SEQ ID NO: 217.

[0133] In certain embodiments, the first binding domain is located N-terminal to the second binding domain.

[0134] In certain embodiments, the second binding domain is located N-terminal to the third binding domain.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. Specifically, the trispecific antibody is an anti-EGFRxCD3xCD28 antibody.

[0136] In another aspect, the present application provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of the preceding.

[0137] In another aspect, the present application provides a vector comprising the nucleic acid molecule of any one of the preceding.

[0138] In certain embodiments, the vector comprises an expression vector. In certain embodiments, the vector comprises a DNA vector and an RNA vector.

[0139] In certain embodiments, the RNA vector is an mRNA vector.

[0140] In another aspect, the present application provides a cell comprising the antibody or antigen binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, and / or the vector of any one of the preceding. Specifically, the cell is obtained by transformation of the vector of any one of the preceding. After transformation of the cell by the vector of any one of the preceding, the antibody or antigen binding fragment thereof of any one of the preceding and / or the nucleic acid molecule of any one of the preceding is expressed in the cell and secreted out of the cell.

[0141] In certain embodiments, the cell comprises a host cell. Specifically, it can be an antibody-producing engineered cell, such as a HEK293 cell.

[0142] In another aspect, the present application provides a method of producing the antibody or antigen binding fragment thereof of any one of the preceding. Specifically, the method comprises:

[0143] (1) constructing an expression vector containing a gene sequence encoding the antibody or antigen binding fragment thereof,

[0144] (2) transforming the expression vector into a host cell to induce expression, and

[0145] (3) isolating the antibody or antigen binding fragment thereof from the expression product.

[0146] In another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or optionally a pharmaceutically acceptable carrier.

[0147] In certain embodiments, the pharmaceutical composition is used as a monotherapy.

[0148] In certain embodiments, the pharmaceutical composition is used in combination with other drugs. Preferably, the other drugs are small molecule targeted anti-cancer agents, other antibody drugs, adoptive cell therapy, and / or oncolytic virus enhancers.

[0149] In another aspect, the present application provides a kit comprising the antibody or antigen binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding.

[0150] In certain embodiments, the kit is used for detecting the content of CD28 in a mixed system.

[0151] In certain embodiments, the kit is used for detecting the content of CD3 in a mixed system.

[0152] In certain embodiments, the kit is used to detect the amount of the corresponding TAA in a mixed system.

[0153] In another aspect, the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding, is used for treating a disease and / or a disorder, alone or in combination with other therapies. In particular, the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding, is used for the manufacture of a medicament for treating a disease.

[0154] In certain embodiments, the disease and / or disorder comprises a tumor. The tumor comprises a solid tumor.

[0155] For example, the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioma, head and neck squamous carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, stomach cancer, thymus cancer, and endometrial cancer.

[0156] In certain embodiments, the disease and / or disorder comprises a tumor. The tumor comprises a non-solid tumor.

[0157] For example, the non-solid tumor comprises a hematological tumor. The hematological tumor comprises leukemia and lymphoma. For another example, the non-solid tumor comprises a hematological tumor. The hematological tumor is selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, myelodysplastic syndrome, and plasmacytoma.

[0158] In certain embodiments, the disease and / or disorder comprises an immunodeficiency disease.

[0159] For example, the immunodeficiency disease is selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, post-surgical complications, and Sjogren-Larsson syndrome.

[0160] Other aspects and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. Only examples of the present application are shown and described in the detailed description. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the application as set forth in the following claims. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0161] The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates are better understood from the detailed description of the exemplary embodiments and drawings following below. The drawings are briefly described as follows:

[0162] Figure 1 shows the configuration schematic of the trispecific antibodies described in this application.

[0163] Figures 2A-2D show the configuration schematic of anti-EGFRxCD3xCD28 antibody, anti-PSMAxCD3xCD28 antibody, anti-5T4xCD3xCD28 antibody and anti-MSLNxCD3xCD28 antibody, respectively, constructed based on the third binding domain that specifically binds to CD28 protected by this application.

[0164] Figures 3A-3D show the configuration schematic of anti-EGFRxCD3xCD28 antibody, anti-PSMAxCD3xCD28 antibody, anti-MSLNxCD3xCD28 antibody and anti-5T4xCD3xCD28 antibody, respectively, constructed based on the anti-CD28 control antibody TGN1412.

[0165] Figure 3E shows the IgG-like configuration trispecific antibodies SAR-SEQ64, SAR-SEQ65, SAR-SEQ61 constructed based on the anti-CD28 control antibody TGN1412 (IgG-like antibody configuration reference: Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86-98 (2020.)).

[0166] Figures 4A-4E show the T cell activation effect mediated by the trispecific antibodies constructed based on the third binding domain that specifically binds to CD28 protected by this application and the control antibody, respectively, at different concentration gradients (Jurkat-NFAT reporter cell system), in which the vertical coordinate represents the fluorescence intensity (RLU). Figure 4A shows the results of anti-PSMAxCD3xCD28 antibody; Figures 4B-4D show the results of anti-MSLNxCD3xCD28 antibody; and Figure 4E shows the results of anti-EGFRxCD3xCD28 antibody.

[0167] FIGS. 5A-5D show the killing activity of human PBMCs mediated by the trispecific antibodies constructed based on the third binding domain specifically binding to CD28 and control antibodies against target cells at different concentration gradients. FIG. 5A shows the results of anti-PSMAxCD3xCD28 antibodies; FIGS. 5B-5C show the results of anti-MSLNxCD3xCD28 antibodies; and FIG. 5D shows the results of anti-EGFRxCD3xCD28 antibodies.

[0168] FIGS. 6A-6E show the photographs of the ovarian cancer organoid model co-incubated with T cells after the trispecific antibodies constructed based on the third binding domain specifically binding to CD28 and control antibodies. FIGS. 6A-6B and FIG. 6D show the results of anti-MSLNxCD3xCD28 antibodies constructed based on the CD28 control antibody at different concentration gradients; FIG. 6C shows the results of anti-MSLNxCD3xCD28 antibodies constructed based on the third binding domain specifically binding to CD28 protected by the present application at different concentration gradients; and FIG. 6E shows the results of the ovarian cancer organoid model cultured with T cells alone or co-incubated.

[0169] FIG. 7 shows the supernatant LDH data of the ovarian cancer organoid model co-incubated with T cells for 120 hours after the anti-MSLNxCD3xCD28 antibodies constructed based on the third binding domain specifically binding to CD28 and control antibodies at different concentration gradients.

[0170] FIGS. 8A-8C show the tumor inhibition effect of the anti-MSLNxCD3xCD28 antibodies constructed based on the third binding domain specifically binding to CD28 and control antibodies in the mouse subcutaneous transplantation of human (HCC1954) breast cancer tumor-bearing model. FIG. 8A shows the dynamic change of the body weight of the mice after administration; FIG. 8B shows the dynamic change of the subcutaneous tumor volume of the mice after administration; and FIG. 8C shows the weight of the subcutaneous tumor removed from the sacrificed mice at the end of the study. DETAILED DESCRIPTION

[0171] The embodiments of the present application are illustrated by the following specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present specification.

[0172] TERMS DEFINITION

[0173] Unless otherwise defined, scientific and technical terms used in this application shall have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used herein and the laboratory procedures are those well- established in the art. Standard techniques can be used for chemical synthesis and chemical analyses.

[0174] In the present application, the term "complementarity determining region (CDR)" refers to amino acid residues of antibody variable domains that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementarity determining region can include, but is not limited to, the amino acid residues from the "complementarity determining regions" as defined by Kabat (i.e., approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Bethesda, Md.: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health (published by the National Institutes of Health, Bethesda, Md., U.S. Dept. of Health and Human Services, Public Health Service), and / or those residues from a "hypervariable loop" (i.e., approximately residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0175] In the present application, the term "antigen binding fragment" generally refers to a polypeptide construct derived from an antibody that is capable of specifically binding an antigen, but which does not comprise all of the elements of a native antibody. In particular, an antigen binding fragment does not comprise some or all of the constant domains of an antibody, and can comprise only one, rather than two, antigen binding sites. In the present application, based on "antigen binding fragment" is generally a polypeptide, so it can also be referred to as "antigen binding polypeptide".

[0176] In the present application, the term "antibody" is interpreted in a broader sense in the context of the present application, i.e. not only comprising traditional IgG class antibodies, but also some other types of antibodies, including but not limited to single chain antibodies, single domain antibodies (VHH), monoclonal antibodies, polyclonal antibodies, dimers, multimers, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies. For example, an IgG class antibody can be an immunoglobulin with a Y-shaped structure that is produced by the immune system in response to a foreign substance, such as a pathogen. A classical structure antibody is a homodimer, each monomer of which can comprise one heavy chain and one light chain connected by disulfide bonds. The light chain consists of a variable region (VL) and a constant region (CL), while the heavy chain consists of a variable region (VH) and three constant regions (CH1, CH2 and CH3). Each variable region contains three "complementarity determining regions" (CDRs), which constitute the antigen binding site responsible for complementarity with an antigen. The antigen binding fragment composed of VL, CL, VH and CH1 is referred to as a "Fab fragment". The "Fab fragment" can be obtained by papain hydrolysis of an antibody, or can be synthesized in vitro. The remaining part, the "trunk" part below the Y-shaped structure, is referred to as the "Fc" region, which is composed of the constant regions CH2 and CH3 domains of the antibody heavy chain. The "hinge region" is the part between CH1 and CH2 in IgG, IgA and IgG class immunoglobulin molecules, which connects the Fab and Fc regions. "F(ab')2" is the product obtained by pepsin hydrolysis of an antibody. F(ab')2 can comprise two Fab fragments and all or part of the hinge region.

[0177] In the present application, the term "specifically binds" generally refers to the binding of an agent, such as an antibody, to a target, such as an epitope, to which it is specific, stronger than to another target. An agent binds to a first target stronger than to a second target if the dissociation constant (Kd) of the agent for the first target is lower than the dissociation constant for the second target. The dissociation constant (Kd) referred to herein is the dissociation constant of the antibody-antigen complex. d ) of the agent for the first target is lower than the dissociation constant for the second target. The dissociation constant (K d) is generally used to reflect the strength of the noncovalent interaction, i.e., the "binding affinity," between a single binding site of a molecule (e.g., an antibody or chimeric antibody immune effector cell engager) and its binding partner (e.g., an antigen). Affinity can be measured by common methods known in the art, including those described in the Examples herein, with low affinity antibodies generally binding antigen slowly and tending to dissociate readily, and high affinity antibodies generally binding antigen more quickly and tending to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present application. Preferably, the dissociation constant for a target to which a reagent specifically binds is more than 100-fold, 200-fold, 500-fold, or more than 1000-fold lower than the dissociation constant for a target to which the reagent does not specifically bind; or, in certain embodiments, an antigen binding polypeptide that specifically binds a target has a dissociation constant (K d ) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. Furthermore, the affinity constant K d , the reciprocal of the dissociation constant K a , can also reflect binding affinity, and can in particular refer to the strength of the antibody and antigen binding. Thus, the term "specifically binds" can also refer to a binding affinity between the binding partners of at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 . An antibody specific for an antigen is in particular an antibody that is capable of binding said antigen with a K a of at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 . a

[0178] In the present application, the term "nucleotide sequence" refers to an order of at least one or more nucleotides linked to each other. The nucleotides can be the main components of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleotides in the nucleotide sequence can be linked in a dehydrated condensation form. The nucleotide sequence described in the present application can be a coding gene of one or more antigen binding polypeptides or fragments thereof.

[0179] ​In the present application, the term "amino acid sequence" refers to an order in which at least one or more amino acids are linked to each other. The amino acid sequence can be a primary structure of a polypeptide or a protein. The amino acids in the amino acid sequence can be linked in a dehydrated condensation form. The amino acid sequence described in the present application can encode one or more antigen-binding polypeptides or fragments thereof.

[0180] In the present application, the term "sequence" needs to be determined in connection with the context, which can refer to "nucleotide sequence" or "amino acid sequence".

[0181] "Amino acid 'conservative substitution'" is well known in the art and generally refers to changing one amino acid residue to another amino acid residue having a structurally or functionally similar side chain. For example, a list of exemplary conservative substitutions is provided in the following table.

[0182] In the present application, the term "variable region" refers to a portion of a single light chain or heavy chain of an antibody molecule comprising CDR and framework region (FR). Among them, the variable region on the light chain is called light chain variable region (VL); the variable region on the heavy chain is called heavy chain variable region (VH).

[0183] In the present application, the term "Fv" refers to a monovalent antigen-binding fragment of a human monoclonal antibody. The Fv fragment can include all or part of the heavy chain variable region (VH) and the light chain variable region (VL), and lacks the heavy chain constant region (CH) and the light chain constant region (CL). The VH and VL can include, for example, CDR. For example, the Fv fragment includes all or part of the amino-terminal variable region of about 110 amino acids of the heavy chain and the light chain.

[0184] In the present application, the term "scFv" refers to a protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain. Among them, the light chain and heavy chain variable regions can be contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed in the form of a single-chain polypeptide, and wherein the scFv can retain the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, the scFv can have the VL and VH variable regions in any order (for example, with respect to the N-terminus and C-terminus of the polypeptide). The scFv can include NH2-VL-linker sequence-VH-COOH, and can also include NH2-VH-linker sequence-VL-COOH.

[0185] In the present application, the term "monoclonal antibody" refers to antibodies produced by a single cell population. In some embodiments, the monoclonal antibody can be produced by hybridoma technology. Hybridoma technology refers to a population of hybrid cells resulting from the fusion of two cells. In some embodiments, the two cells can be a B cell, which provides the plasma cell to make the corresponding antibody, and a tumor cell, which has the ability of unlimited proliferation. By combining the characteristics of both, a hybridoma cell with the ability of unlimited proliferation to produce antibodies is formed. In the present application, the term "polyclonal antibody" refers to antibodies produced by a plurality of cell populations. In some embodiments, the polyclonal antibody can be obtained by injecting a purified antigen into an animal and purifying the antibody from the serum of the animal. The "monoclonal antibody" and the "polyclonal antibody" recognize the same antigen, but differ in that the "monoclonal antibody" is produced by a single cell population, while the "polyclonal antibody" is produced by a plurality of cell populations.

[0186] In the present application, the term "monospecific antibody" refers to an antibody having one or more binding sites, each of which has the same binding specificity, i.e., binds to the same antigen or hapten. The "monospecific antibody" can be a "monoclonal antibody" or a "polyclonal antibody". In the present application, the term "multispecific antibody" refers to an antibody having two or more variable regions that bind to different antigens. The "monospecific antibody" and the "multispecific antibody" differ in that the "monospecific antibody" can bind to a single or multiple epitopes, but these epitopes are all from the same antigen; the "multispecific antibody" binds to two or more epitopes, and these epitopes are from two or more antigens. In the examples of the present application, the "multispecific antibody" can be a "bispecific antibody" or a "trispecific antibody".

[0187] In the present application, the term "bispecific antibody" refers to an antibody that specifically binds to two antigens. In the present application, the configuration of the "bispecific antibody" can be arbitrary. Taking the anti-CD3xCD28 antibody as an example, the configuration of the antibody can be a single chain that does not include a constant region connected by a linker, such as VH CD3 - linker - VL CD3 - linker - VH CD28 - linker - VL CD28 or VH CD3 - linker - VL CD28 - linker - VH CD28 - linker - VL CD3The configuration of the antibody can also be a plurality of chains not containing the constant region, each chain can be connected by covalent bond such as disulfide bond or other ways; the configuration of the antibody can also be a single chain containing the constant region; the configuration of the antibody can also be a plurality of chains containing the constant region and other configurations capable of exercising the function of the antibody.

[0188] In the present application, the term "trispecific antibody" refers to an antibody that specifically binds three antigens. Similar to "bispecific antibody", "trispecific antibody" also has arbitrariness in configuration, and the specific possible configurations can be referred to the above "bispecific antibody".

[0189] In the present application, the "linker sequence" refers to a peptide segment for connecting two binding domains, and also refers to a peptide segment for connecting VH and VL in a binding domain. According to the location of the "linker sequence", it can be divided into "intra-domain linker" and "inter-domain linker", the "intra-domain linker" refers to a peptide segment for connecting VH and VL in a binding domain; the "inter-domain linker" refers to a peptide segment for connecting two binding domains. In the antibody or antigen binding fragment thereof with 3 binding domains described in the present application, the "first inter-domain linker" specifically refers to a linker for connecting the first binding domain and the second binding domain, and the "second inter-domain linker" specifically refers to a linker for connecting the second binding domain and the third binding domain.

[0190] In the present application, the term "binding domain" refers to a polypeptide structure capable of specifically recognizing and binding to a target in the context of a multispecific antigen binding polypeptide, sometimes also referred to as "domain".

[0191] In the present application, the term "T cell engager (TCE)" generally refers to a class of artificial bispecific or multispecific antibodies based on antibodies or antibody fragments, which can connect T cells in vivo with cells expressing specific tumor target related proteins. In some embodiments, the TCE is a bispecific antibody, also known as "bispecific T cell engager" or BiTE (Bispecific T-cell Engager), which has two different target antigen binding domains, one side of which can recognize tumor related antigens (such as CD19, CD33, BCMA, etc.), and the other side is usually specific to CD3 / T cell receptor (TCR) complex, so as to engage T cells and tumor target cells, cause T cell activation and kill tumor target cells. The term "CD3 +TCE” generally refers to an antibody that engages CD3 on T cell surface and a specific antigen on tumor cell surface, which can recruit and activate polyclonal T cell population near the tumor and kill the tumor cells. However, in some cases, two activation signals are required for the initial T lymphocyte to go from quiescence to full activation. This is because the signal transmitted by CD3-TCR complex is not enough to fully activate T cells after the antigen / MHC complex is recognized by TCR, and the signal generated by the costimulatory molecule is needed as a supplement. As used herein, “costimulatory CD3 + TCE” refers to a multispecific antibody, usually trispecific antibody, that simultaneously achieves targeting of tumor cells and “double signal” activation of T cells (the first signal comes from CD3 / TCR recognizing MHC / antigen peptide complex; the second signal is provided by a costimulatory molecule, which is a costimulatory signal). For example, B7-CD28 complex formed by the non-covalent binding of CD28 molecule and ligand (B7) on antigen presenting cells (APC) can synergize with CD3 antibody to exert costimulatory effect, stimulate T cell differentiation, proliferation and secretion of cytokines such as IL-2, and play an important role in tumor immunity, autoimmune disease treatment and immune intervention against transplant rejection.

[0192] In the present application, the term “single-chain antibody” refers to an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In contrast to “scFv”, “single-chain antibody” can independently perform antibody functions, while scFv may or may not be able to independently perform antibody functions.

[0193] In the present application, the term “single-domain antibody (VHH)” refers to a fragment containing a single variable domain of an antibody, which can selectively bind to a specific antigen. The “single-domain antibody” can contain a heavy chain variable region. In comparison with intact antibodies or scFv / Fv fragments, the single-domain antibody has a lower molecular weight and is more easily permeable into tissues. In some embodiments, the single-domain antibody can be isolated from llama serum.

[0194] In the present application, the term “dimer” refers to the same or similar substances in a double form. The “double form” refers to two molecules each having its own function to form a new molecule. The “dimer” can be two monomers connected by a chemical bond. In the present application, the term “multimer” refers to a molecule formed by the connection of two or more monomers by a chemical bond. In some embodiments, “dimer” and “multimer” refer to a high molecular complex formed by the combination of multiple protein molecules, which can have properties or functions that are not present in a single state.

[0195] In the present application, the term "human antibody" refers to an antibody whose variable and constant regions are derived entirely from human germline immunoglobulin sequences. All sequences of a "human antibody" are derived from human germline and do not comprise any heterologous germline sequences.

[0196] In the present application, the term "humanized antibody" refers to an antibody in which the framework or "complementarity determining regions (CDRs)" have been modified to include immunoglobulin CDRs of different specificity compared to the CDRs of the parent immunoglobulin. For example, mouse CDRs are grafted into the framework region of a human antibody to make the "humanized antibody". The "humanized antibody" has CDR sequences of a heterologous germline compared to the "human antibody", but its framework is still of human germline.

[0197] In the present application, the term "chimeric antibody" generally refers to a monoclonal antibody that includes variable regions from one species origin and at least a portion of a constant region from another species origin. "Chimeric antibodies" can be made by recombinant DNA techniques. For example, a chimeric antibody that includes murine variable regions and human constant regions. Such murine / human chimeric antibodies are the product of an immunoglobulin gene expressed from a DNA segment that includes a DNA segment encoding murine immunoglobulin variable regions and a DNA segment encoding human immunoglobulin constant regions. The sequence of the chimeric antibody comprises a heterologous germline compared to the "human antibody". The chimeric antibody is directed to a longer variable region than the CDR region compared to the "humanized antibody", which is greater than "humanized antibodies" on the recombination unit.

[0198] In the present application, the term "T cell surface molecule" refers to a molecule expressed on the cell membrane of a T cell. Specifically, the molecule can be a protein (including but not limited to glycoprotein and lipoprotein), a polysaccharide, or a lipid. In the examples of the present application, "T cell surface molecules" include but are not limited to CD3, CD4 or CD8.

[0199] In the present application, the term "CD3" generally refers to T cell co-receptor CD3 (cluster of differentiation 3), which is a transmembrane protein, and its transmembrane region is connected to the transmembrane region of the two peptide chains of TCR through a salt bridge, forming a TCR-CD3 complex, which participates in the recognition of T cells to antigens and transduces the activation signal generated to the T cells. Without special instructions, the second binding domain specifically binds to CD3 in the context of the present application. It is usually composed of four different polypeptide chains, CD3γ chain, CD3δ chain and two CD3ε chains. Together with the T cell receptor (TCR), CD3 can form a TCR-CD3 complex.

[0200] In the present application, the term "CD28" generally refers to a dimeric transmembrane glycoprotein, which is expressed on approximately 80% of human CD4 + T cells and 50% of CD8+ T cell surface expression. CD28 is a T cell costimulatory molecule that binds to the ligands CD80 (B7-1) or CD86 (B7-2) and amplifies the primary signal of TCR / CD3 conduction, maintains T cell survival, and promotes cytokine-induced T cell proliferation and differentiation. Unless otherwise specified, the CD28 to which the third binding domain specifically binds in the context of the present application is human CD28.

[0201] In the present application, the term "cross-reactivity" refers to the ability of an antibody that specifically binds to one antigen to bind to another antigen. This cross-reactivity can be referred to as "intraspecies cross-reactivity" or "inter-target cross-reactivity" if the two antigens are from the same species. This cross-reactivity is referred to as "interspecies cross-reactivity" if the two antigens are from different species. Unless otherwise specified, "cross-reactivity" of an antibody in the context of the present application refers to interspecies cross-reactivity, i.e. the ability to react with homologous or orthologous proteins derived from other species.

[0202] In the present application, the term "nucleic acid" or "isolated nucleic acid molecule" can include single- and double-stranded nucleic acids and ribonucleic acids as well as deoxyribonucleic acids. It can comprise naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified, e.g. by methylation, 5'- and / or 3'-capping. In the present application, the term "nucleic acid encoding for... " or "(nucleotide) sequence encoding for... " generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes for a protein. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and a polyadenylation signal capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered. The coding sequence can be codon-optimized.

[0203] In the present application, the term "vector" or "expression vector" generally refers to any molecule (e.g., nucleic acid, plasmid or virus, virus-like particle, polycation, peptide vector, liposome, and / or hybrid vector) 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 "plasmid," which refers to a circular double stranded DNA molecule onto which additional DNA segments can be inserted. Another type of vector is a virus vector, wherein additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Additionally, in some instances, "liposomes" or "lipid nanoparticles (LNP)" are also commonly used to deliver DNA molecules and / or RNA molecules (e.g., DNA or mRNA encoding an antigen-binding polypeptide of the present application). Among others, liposomes generally refer to a vesicle having an interior space that is sequestered from the external milieu by a membrane or membranes of one or more bilayers, which are biocompatible, non-toxic, can therefore deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB). In certain instances, LNP can also be used to deliver RNP complexes. LNP generally refers to a particle comprising multiple (i.e., more than one) lipid molecules that are physically associated (e.g., covalently or non-covalently) with each other by intermolecular forces; for example, when LNP is used to deliver a nucleic acid molecule of the present application, it can encapsulate the nucleic acid within a cationic lipid particle (e.g., a liposome), and can relatively easily deliver the nucleic acid molecule to a cell. Other vectors (e.g., non-episomal mammalian vectors) can

[0204] In the present application, the term "cell" and "host cell" are used interchangeably and generally refer to a cell that comprises exogenous recombinant DNA, such as an expression vector, and also to a cell that comprises a polypeptide or protein obtained by expression from the recombinant DNA. Such host cells can be used to produce the trispecific antibodies of the present application. In certain embodiments, as a host cell, there is no particular limitation as long as it is a host cell that can be used for expression of a polypeptide sequence, such as the trispecific antibodies of the present application. A recombinant host cell or host cell means not only the particular subject cell, but also its progeny. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny can not, in principle, be identical to the parent cell, but such cells are still included within the scope of the term "cell" or "host cell" as used herein.

[0205] In the present application, the term "kit" generally refers to a combination of reagents and other materials. A kit can comprise reagents such as buffers, protein stabilizing reagents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test vessels (e.g., microtiter plates, etc.). The term "kit" is not intended to be limited to a particular combination of reagents and / or other materials. For example, a kit can also comprise instructions for using the reagents. A test kit can be packaged in any suitable manner, typically, it has the components in a single container or, if necessary, in multiple containers, and a sheet of instructions for performing the test. For example, a kit can also include a positive control sample. A kit can be prepared in a variety of ways known in the art.

[0206] In the present application, the term "mixture" refers to a product comprising any amount of any ingredient, and any product produced directly or indirectly from a combination of any amount of any ingredient. A "mixture" can exist in any form, such as a suspension, such as an aerosol; a "mixture" can also be a sample isolated from an organism, such as blood, urine, etc. In certain embodiments, a "mixture" can or can not comprise CD28. In contrast to terms such as "reagent", the ingredients and amounts of a "mixture" are not specified, and can or can not be specified.

[0207] In the present application, the term "drug" or "pharmaceutical composition" generally refers to a compound or composition that is capable of inducing a desired therapeutic effect when appropriately administered to a patient. Preferably, a pharmaceutical composition comprises a drug of the present application (e.g., an antigen-binding polypeptide or a nucleic acid molecule) or a variant, prodrug, or other biologically active effective form thereof, and a carrier, diluent, or pharmaceutical excipient, such as a buffer, a preservative, and a tonicity adjusting agent.

[0208] In the present application, the term "pharmaceutically acceptable carrier" generally refers to one or more formulation materials suitable for effecting or enhancing delivery of a binding protein.

[0209] In the present application, the terms "effective amount" and "therapeutically effective amount" are used interchangeably and generally refer to an amount or dose of a pharmaceutical composition comprising one or more binding proteins that is sufficient to produce a desired therapeutic result when administered. More specifically, a therapeutically effective amount is an amount of a drug (e.g., an antigen binding polypeptide provided herein) or a pharmaceutical composition thereof that is sufficient to treat a specified condition, disorder, or disease, particularly to ameliorate, alleviate, relieve, and / or delay one or more symptoms thereof, for a period of time. In some examples directed to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or other unwanted proliferating cells. In some embodiments, an effective amount is an amount sufficient to delay the development of a tumor or cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay the recurrence of a tumor or cancer. An effective amount can be administered in one or more administrations. In some embodiments, an effective amount of a drug or a pharmaceutical composition thereof can: (1) reduce the number of cancer cells; (2) reduce tumor size; (3) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (4) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (5) inhibit tumor growth; (6) prevent or delay occurrence and / or recurrence of a tumor; (7) relieve to some extent one or more of the symptoms associated with the cancer. The effective amount can vary depending on the specific antigen binding polypeptide being used and also depending on a variety of factors and conditions related to the patient being treated and the severity of the condition. For example, if the antigen binding polypeptide is to be administered in vivo, factors such as the age, body weight, and health of the patient and dose response curves and toxicity data obtained in preclinical animal work would be among those considered. Determining an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0210] In the present application, the term "treatment" generally refers to therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include patients who are suffering from a condition as well as patients who are in danger of developing a condition or who are to be prevented from developing a condition. In particular embodiments, the binding proteins can be used to treat a human who has cancer, or a human who is predisposed to cancer, or to ameliorate cancer in a human subject. The binding proteins can also be used to prevent cancer in a human patient.

[0211] In the present application, the term "immunodeficiency disease" refers to a disease caused by a malfunction or deficiency in the ability of the immune system to fight disease. The immunodeficiency disease can be primary (such as congenital immunodeficiency due to abnormal expression of certain genes) or secondary (such as abnormal immune function due to viral infection). The immunodeficiency disease can be abnormally active (such as systemic lupus erythematosus) or abnormally low (such as AIDS). It is worth noting that, according to the usual understanding of those skilled in the art, cancer should also belong to one of the immunodeficiency diseases, but in order to better distinguish different concepts, the present application separates cancer from immunodeficiency diseases, i.e. the immunodeficiency diseases described in the present application do not include cancer, but include other immunodeficiency diseases in addition to cancer.

[0212] In the present application, the term "tumor" or "cancer" refers to cells with the ability to grow autonomously. Examples of the cells include, but are not limited to, cells with abnormal states or conditions characterized by rapid proliferation of cell growth. This term can include cancer growth, such as tumors, carcinogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasion. It can also include malignant tumors of various organ systems (such as respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system) and adenocarcinoma (such as most colon cancer, renal cell carcinoma, prostate cancer, and / or testicular tumor, non-small cell lung cancer, and small intestine cancer). The tumor or cancer can be naturally occurring or artificially induced by experimental means (the subject is an ethically acceptable experimental animal). The "naturally occurring" cancer can include any cancer induced by experimental means, including but not limited to spontaneously occurring cancer, cancer caused by exposure of the patient to carcinogens, cancer caused by insertion of oncogenes or knockout of tumor suppressor genes, or cancer caused by infection (e.g. viral infection).

[0213] In the present application, the term "patient" can generally include a human, a non-human primate (e.g., a monkey), or other animals, particularly mammals, such as a cow, a horse, a pig, a sheep, a goat, a dog, a cat, or a rodent, such as a mouse and a rat. In particularly preferred embodiments, the patient is a human.

[0214] In the present application, the term "and / or" should be understood as a combination of any one of the elements or any combination of several elements connected by the term.

[0215] In the present application, the term "comprising" or including generally means including the explicitly specified features, but not excluding other elements.

[0216] In the present application, the term "selected from" generally refers to the inclusion of the selected objects and all combinations thereof. For example, "selected from A, B, and C" means to include all combinations of A, B, and C, e.g., A, B, C, A+B, A+C, B+C, or A+B+C.

[0217] In the present application, the term "about" generally refers to a variation within a range of 0.5-10% above or below the designated numerical value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the designated numerical value.

[0218] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use in the present application; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0219] DETAILED DESCRIPTION

[0220] Those skilled in the art will appreciate that the determination of CDR regions can be determined using any of the Kabat, IMGT, Chothia antibody numbering systems. When different numbering systems are used, the amino acid sequences for the same variable region can demarcate different CDR regions. CDR regions determined using either numbering system fall within the scope of the present application. Unless otherwise indicated, the specific CDR region amino acid sequences recited in the present application are determined according to the Kabat antibody numbering system.

[0221] Antibodies or antigen-binding fragments thereof that specifically bind CD28

[0222] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds a costimulatory molecule. In specific embodiments, the costimulatory molecule can be CD28, and the antibody or antigen-binding fragment thereof comprises a domain that specifically binds CD28. Preferably, the binding is to human CD28.

[0223] The antibodies or antigen-binding fragments thereof that specifically bind to human CD28 protected by the present application have advantages in functions, specifically, they have (1) high affinity to CD28 target antigen; (2) promote the binding of TAA binding domain to its target antigen; (3) induce stronger T cell activation and T cell proliferation differentiation ability; (4) mediate more effective tumor killing and antitumor effect in vivo. In preferred embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to human CD28 protected by the present application have an affinity of less than 0.5 nM.

[0224] In certain embodiments, the domain that specifically binds to CD28 can comprise one, two or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13; and / or, one, two or three light chain variable region CDRs selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 25 and SEQ ID NO: 31.

[0225] In certain embodiments, the domain that specifically binds to CD28 can comprise:

[0226] (1) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), a heavy chain complementarity determining region 3 (HCDR3); and

[0227] (2) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), a light chain complementarity determining region 3 (LCDR3).

[0228] The HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 1. The HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 7. The HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 13. The LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 19. The LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 25. The LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 31.

[0229] For example, the VH can comprise an HCDR1 as set forth in SEQ ID NO: 1, an HCDR2 as set forth in SEQ ID NO: 7, and an HCDR3 as set forth in SEQ ID NO: 13; and the VL can comprise an LCDR1 as set forth in SEQ ID NO: 19, an LCDR2 as set forth in SEQ ID NO: 25, and an LCDR3 as set forth in SEQ ID NO: 31.

[0230] In certain embodiments, the antibody or antigen-binding fragment thereof can be a scFv that specifically binds to CD28.

[0231] In further preferred embodiments of the above-mentioned embodiments, the scFv that specifically binds to CD28 can comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0232] (1) the VH can comprise an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 37 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0233] the VL can comprise an amino acid sequence as set forth in SEQ ID NO: 48, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 48 and all amino acid differences are in the amino acid sequence of the non-CDR regions; or

[0234] (2) the VH can comprise an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 38 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0235] the VL can comprise an amino acid sequence as set forth in SEQ ID NO: 49, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 49 and all amino acid differences are in the amino acid sequence of the non-CDR regions.

[0236] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0237] In certain embodiments, in the scFv specifically binding to CD28, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL can be a first linker sequence or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer of 1-10, preferably an integer of 1-5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv specifically binding to CD28, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0238] In certain embodiments, the antibody specifically binding to CD28 can be a monospecific antibody specifically binding to CD28.

[0239] In certain embodiments, the antibody specifically binding to CD28 can be a single chain antibody specifically binding to CD28. The single chain antibody can comprise any of the foregoing scFv specifically binding to CD28 and necessary components.

[0240] In certain embodiments, the antibody specifically binding to CD28 can be a conventional antibody specifically binding to CD28, preferably, can be an IgG class antibody. The conventional antibody can comprise any of the foregoing VH portion, VL portion of the domain specifically binding to CD28 and necessary linker sequence, constant region components.

[0241] Antibody or antigen-binding fragment thereof specifically binding to CD28 and CD3

[0242] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to CD28, which can comprise the domain that specifically binds to CD28 of any of the foregoing. The antibody or antigen-binding fragment thereof can further comprise a domain that specifically binds to another antigen. In certain embodiments, the other antigen can be a T cell surface molecule. Preferably, the T cell surface molecule can be selected from the group consisting of CD2, CD3, CD4, CD8, CD40, CD137, OX40 and GITR. In specific embodiments, the T cell surface molecule can be CD3, preferably human CD3, and the antibody or antigen-binding fragment thereof comprises a domain that specifically binds to CD3.

[0243] The antibody or antigen-binding fragment thereof that specifically binds to human CD28 and human CD3 protected by the present application has advantages in function, in particular, it has higher binding activity and stronger tumor killing ability by itself and downstream products constructed based thereon, and in particular, it should also have stronger ability to induce T cell proliferation and differentiation.

[0244] In certain embodiments, the domain that specifically binds to CD3 can comprise one, two or three heavy chain variable region CDRs selected from the amino acid sequence set forth in SEQ ID NO: 205; and / or, one, two or three light chain variable region CDRs selected from the amino acid sequence set forth in SEQ ID NO: 206.

[0245] For example, the domain that specifically binds to CD3 can comprise HCDR1, HCDR2 and HCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 205; and LCDR1, LCDR2 and LCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 206.

[0246] In certain embodiments, the domain that specifically binds to CD3 can be a scFv that specifically binds to CD3. The scFv sequence that specifically binds to CD3 can be derived from an anti-CD3 antibody or antibody analog, derivative known in the art, respectively. For example, the scFv can be a scFv comprising VH and VL derived from a known corresponding antibody.

[0247] In further preferred embodiments of the foregoing embodiments, the scFv that specifically binds to CD3 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Among them

[0248] (1) the VH can comprise an amino acid sequence as set forth in SEQ ID NO: 205, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 205 and all amino acid differences are in non-CDR regions; and

[0249] (2) the VL can comprise an amino acid sequence as set forth in SEQ ID NO: 206, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 206 and all amino acid differences are in non-CDR regions.

[0250] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0251] In certain embodiments, in the scFv that specifically binds to CD3, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and the VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and the VL in the scFv that specifically binds to CD3 can be a first linker sequence, can be a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds to CD3, the intradomain linker between the VH and the VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0252] In certain embodiments, in the antibody or antigen-binding fragment thereof, the scFv specifically binding to CD28 and the scFv specifically binding to CD3 can further comprise a linker sequence, particularly an interdomain linker, between them. In certain embodiments, the interdomain linker can be the first linker sequence, can be the second linker sequence. In preferred embodiments, the interdomain linker can be the second linker sequence. In certain embodiments, the second linker sequence can be a linker sequence from the IgD hinge region. In particular embodiments, the second linker sequence can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219, preferably SEQ ID NO: 217.

[0253] In certain embodiments, the antibody or antigen-binding fragment thereof can be a bispecific antibody specifically binding to CD28 and CD3, also referred to herein as anti-CD3xCD28 antibody.

[0254] In certain embodiments, the bispecific antibody can comprise a scFv specifically binding to CD28 and a scFv specifically binding to CD3. In certain embodiments, the scFv specifically binding to CD28 can be located at the N-terminus or C-terminus of the scFv specifically binding to CD3. In certain embodiments, the bispecific antibody can further comprise an interdomain linker, preferably the second linker sequence, between the two scFv. In particular embodiments, the second linker sequence can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219, preferably SEQ ID NO: 217.

[0255] Further, the bispecific antibody can comprise, in order from N-terminus to C-terminus, a scFv specifically binding to CD3, an interdomain linker, a scFv specifically binding to CD28. It can specifically result in the following combinations of domain connections: (1) VL CD3 - intradomain linker - VH CD3 - interdomain linker - VH CD28 - intradomain linker - VL CD28 ; (2) VL CD3 - intradomain linker - VH CD3 - interdomain linker - VH CD28 - intradomain linker - VL CD28 ; (3) VH CD3 - intradomain linker - VL CD3 - interdomain linker - VL CD28 - intradomain linker - VH CD28; or (4) VL CD3 - intra-domain linker - VH CD3 - inter-domain linker - VL CD28 - intra-domain linker - VH CD28 .

[0256] In certain embodiments, the bispecific antibody can further comprise a constant region.

[0257] An antibody or antigen-binding fragment thereof that specifically binds to a TAA

[0258] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to a tumor-associated antigen (TAA). The TAA can be a protein that is expressed by tumor cells but not normal cells, or a protein that is expressed in higher amounts by tumor cells than by normal cells, preferably a cell membrane protein. In specific embodiments, the TAA can be selected from the group consisting of 5T4, B7-H3, BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD46, CD70, CD123, CDH3, CDH6, CDH17, CEACAM5, Claudin 1, Claudin 3, Claudin 6, Claudin 18.2, EGFR, EGFRvIII, ENPP3, folate receptor alpha, GD2, GPC3, Her2, Her3, Her4, IL13ra2, LIV1 (ZIP6), MSLN, MUC1, MUC2, MUC4, MUC5AC, MUC6, MUC16, MUC20, NCAM, Nectin4, NY-ESO-1, PSMA (FOLH1), PSCA, P-cadherin, STEAP-1, SLC44A4, SLITRK6, and TROP2. Preferably, the TAA is selected from the group consisting of PSMA, MSLN, 5T4, and EGFR.

[0259] In specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a TAA can comprise a light / heavy chain variable region sequence that has been humanized in a non-CDR region. The humanization can be performed by any method known in the art that can achieve humanization, such as CDR grafting. The purpose of the humanization is to improve the binding or functional properties of the antibody or antigen-binding fragment thereof disclosed in the prior art by amino acid variation in the non-CDR region, so as to obtain an anti-TAA antibody or antigen-binding fragment thereof with higher affinity and better tumor-killing effect.

[0260] In the present application, the antibodies specifically binding to the respective TAAs, the inventors, although taking the CDR sequences disclosed in the prior art as reference, have correspondingly obtained variable region sequences with better effects by humanizing the non-CDR regions, and hope to obtain protection based on these modified variable region sequences.

[0261] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binding to a TAA can be an antibody or antigen-binding fragment thereof comprising a constant region. In this embodiment, the anti-TAA antibody is preferably a humanized antibody.

[0262] In certain embodiments, the antibody or antigen-binding fragment thereof can be an scFv.

[0263] In certain embodiments, especially when the scFv specifically binding to a TAA is part of a multispecific antibody comprising an anti-CD28 antigen-binding fragment of the present application, the anti-TAA scFv can be derived from an anti-corresponding TAA antibody or antibody analog, derivative known in the art. For example, the scFv can be an scFv comprising a VH and a VL derived from a known corresponding antibody. The antibody can be an animal-derived antibody such as a murine-derived antibody, a chimeric antibody such as a human-murine chimeric antibody, or a human antibody or a humanized antibody.

[0264] PSMA

[0265] In some specific embodiments, the TAA is PSMA, and the antibody or antigen-binding fragment thereof comprises a domain specifically binding to PSMA. The antibody or antigen-binding fragment thereof specifically binding to PSMA can be used for the production of a medicament for treating a PSMA-positive disease, preferably a tumor (such as prostate cancer).

[0266] In certain embodiments, the domain specifically binding to PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Among them

[0267] (1) the VH PSMA may comprise an HCDR1 as shown in SEQ ID NO: 4, an HCDR2 as shown in SEQ ID NO: 10, SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223, and an HCDR3 as shown in SEQ ID NO: 16; and

[0268] (2) the VL PSMA may comprise an LCDR1 as shown in SEQ ID NO: 22 or SEQ ID NO: 224, an LCDR2 as shown in SEQ ID NO: 28, and an LCDR3 as shown in SEQ ID NO: 34.

[0269] For example, the domain that specifically binds PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0270] (1) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 10, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0271] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0272] (2) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 221, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0273] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0274] (3) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 222, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0275] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0276] (4) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 223, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0277] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0278] (5) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 10, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0279] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0280] (6) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 221, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0281] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0282] (7) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 222, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0283] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34; or

[0284] (8) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 223, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0285] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34.

[0286] In further preferred embodiments of the above embodiments, the domain that specifically binds PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0287] (1) The VH PSMA It may contain an amino acid sequence as shown in any one of SEQ ID NO:44 or SEQ ID NO:238-241, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) with all amino acid differences located in the non-CDR region; and

[0288] (2) The VL PSMA It may contain amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:242 or SEQ ID NO:243, or amino acid sequences that have at least 80% homology with the amino acid sequences shown in SEQ ID NO:54, SEQ ID NO:242 or SEQ ID NO:243 (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) and all amino acid differences are in the non-CDR region.

[0289] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0290] In a further preferred embodiment, the PSMA-specific binding domain may include a heavy chain variable region (VH) and a light chain variable region (VL).

[0291] (1) The VH PSMA It may contain an amino acid sequence as shown in any one of SEQ ID NO:44 or SEQ ID NO:238-241; and

[0292] (2) The VL PSMA It may contain amino acid sequences such as those shown in SEQ ID NO:54, SEQ ID NO:242 or SEQ ID NO:243.

[0293] Preferably, the PSMA-specific binding domain may comprise a heavy chain variable region (VH) and a light chain variable region (VL).

[0294] (1) The VH PSMA It may contain an amino acid sequence as shown in SEQ ID NO:238, and VL PSMA It may contain an amino acid sequence as shown in SEQ ID NO:242;

[0295] (2) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 238, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0296] (3) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 44, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0297] (4) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 44, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 243;

[0298] (5) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 239, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0299] (6) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 240, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0300] (7) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 241, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0301] (8) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 239, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 243;

[0302] (9) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 240, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 243; or

[0303] (10) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 241, and the VL PSMAmay comprise an amino acid sequence as set forth in SEQ ID NO: 243.

[0304] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds PSMA can be an antibody or antigen-binding fragment thereof comprising a constant region. In such embodiments, the anti-PSMA antibody is preferably a humanized antibody.

[0305] In certain embodiments, the antibody or antigen-binding fragment thereof can be an scFv.

[0306] In certain embodiments, in the scFv that specifically binds PSMA, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv that specifically binds PSMA can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds PSMA, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0307] In certain embodiments, the antibody that specifically binds PSMA can be a monospecific antibody that specifically binds PSMA.

[0308] In certain embodiments, the antibody that specifically binds PSMA can be a single-chain antibody that specifically binds PSMA. The single-chain antibody can comprise any of the foregoing scFv that specifically binds PSMA and necessary components.

[0309] In certain embodiments, the antibody that specifically binds PSMA can be a conventional antibody that specifically binds PSMA, preferably, an IgG class antibody. The conventional antibody can comprise any of the foregoing VH portion, VL portion, and necessary linker sequence, constant region components of the domain that specifically binds PSMA.

[0310] In certain embodiments, the antibody that specifically binds PSMA can be a multispecific antibody that specifically binds PSMA.

[0311] In certain embodiments, the multispecific antibody can be a bispecific antibody. For example, it can be an anti-PSMAxCD3 antibody or an anti-PSMAxCD28 antibody. The anti-PSMAxCD3 antibody can comprise the domain that specifically binds CD3 of any one of the foregoing; the anti-PSMAxCD28 antibody can comprise the domain that specifically binds CD28 of any one of the foregoing.

[0312] In certain embodiments, the multispecific antibody can be a trispecific antibody.

[0313] MSLN

[0314] In some specific embodiments, the TAA is MSLN, and the antibody or antigen-binding fragment thereof comprises a domain that specifically binds MSLN. The antibody or antigen-binding fragment thereof that specifically binds MSLN can be used for the production of a medicament for treating a MSLN-positive disease, preferably a tumor (such as mesothelioma).

[0315] In certain embodiments, the domain that specifically binds MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0316] (1) the VH can comprise a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 9, and a HCDR3 as set forth in SEQ ID NO: 15; and

[0317] (2) the VL can comprise a LCDR1 as set forth in SEQ ID NO: 21, a LCDR2 as set forth in SEQ ID NO: 27, and a LCDR3 as set forth in SEQ ID NO: 33.

[0318] In further preferred embodiments of the foregoing embodiments, the domain that specifically binds MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0319] (1) The VH may comprise an amino acid sequence as shown in any one of SEQ NO ID:40-43 or SEQ ID NO:45, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) with all amino acid differences in the non-CDR region; and

[0320] (2) The VL may contain an amino acid sequence as shown in any one of SEQ NO ID:51-53 or SEQ ID NO:55, or an amino acid sequence that has at least 80% homology with any one of SEQ NO ID:51-53 or SEQ ID NO:55 (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) and all amino acid differences are in the non-CDR region.

[0321] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0322] In a further preferred embodiment, the domain that specifically binds to MSLN may include a heavy chain variable region (VH) and a light chain variable region (VL).

[0323] (1) The VH may contain an amino acid sequence as shown in any one of SEQ NO ID:40-43 or SEQ ID NO:45; and

[0324] (2) The VL may contain an amino acid sequence as shown in any one of SEQ NO ID:51-53 or SEQ ID NO:55.

[0325] Preferably, the domain that specifically binds to MSLN may include a heavy chain variable region (VH) and a light chain variable region (VL).

[0326] (1) The VH MSLN It may contain an amino acid sequence as shown in SEQ ID NO:40, and VL MSLN It may contain an amino acid sequence as shown in SEQ ID NO:51;

[0327] (2) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:40. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:52;

[0328] (3) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:40. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:53;

[0329] (4) The VH MSLN It may contain an amino acid sequence as shown in SEQ ID NO:41, and VL MSLN It may contain an amino acid sequence as shown in SEQ ID NO:51;

[0330] (5) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:41. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:52;

[0331] (6) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:41. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:53;

[0332] (7) The VH MSLN It may contain an amino acid sequence as shown in SEQ ID NO:42, and VL MSLN It may contain an amino acid sequence as shown in SEQ ID NO:51;

[0333] (8) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:42. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:52;

[0334] (9) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:42. MSLN It may contain an amino acid sequence as shown in SEQ ID NO:53;

[0335] (10) The VH MSLN It may contain an amino acid sequence as shown in SEQ ID NO:43, and VL MSLN It may contain an amino acid sequence as shown in SEQ ID NO:51;

[0336] (11) The VH MSLN The VL may contain an amino acid sequence as shown in SEQ ID NO:43.MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 52; or

[0337] (12) the VH MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 43, and the VL MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 53.

[0338] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to MSLN can be an antibody or antigen-binding fragment thereof comprising a constant region. In such embodiments, the anti-MSLN antibody is preferably a humanized antibody.

[0339] In certain embodiments, the antibody or antigen-binding fragment thereof can be an scFv.

[0340] In certain embodiments, in the scFv that specifically binds to MSLN, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and the VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and the VL in the scFv that specifically binds to MSLN can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer of 1-10, preferably an integer of 1-5, for example 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds to MSLN, the intradomain linker between the VH and the VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0341] In certain embodiments, the antibody that specifically binds to MSLN can be a monospecific antibody that specifically binds to MSLN.

[0342] In certain embodiments, the antibody that specifically binds to MSLN can be a single-chain antibody that specifically binds to MSLN. The single-chain antibody can comprise any of the foregoing scFv that specifically binds to MSLN and necessary components.

[0343] In certain embodiments, the antibody specifically binding to MSLN can be a conventional antibody specifically binding to MSLN, preferably, can be an IgG class antibody. The conventional antibody can comprise the VH portion, the VL portion and the necessary linker sequence, the constant region component of any one of the aforementioned domains specifically binding to MSLN.

[0344] In certain embodiments, the antibody specifically binding to MSLN can be a multispecific antibody specifically binding to MSLN.

[0345] In certain embodiments, the multispecific antibody can be a bispecific antibody. For example, can be an anti-MSLNxCD3 antibody or an anti-MSLNxCD28 antibody. The anti-MSLNxCD3 antibody can comprise the domain specifically binding to CD3 of any one of the aforementioned; the anti-MSLNxCD28 antibody can comprise the domain specifically binding to CD28 of any one of the aforementioned.

[0346] In certain embodiments, the multispecific antibody can be a trispecific antibody.

[0347] 5T4

[0348] In some specific embodiments, the TAA is 5T4, the antibody or antigen binding fragment thereof comprises a domain specifically binding to 5T4. The antibody or antigen binding fragment thereof specifically binding to 5T4 can be used for producing a medicament for treating a 5T4 positive disease, preferably a tumor.

[0349] In certain embodiments, the domain specifically binding to 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0350] (1) the VH 5T4 may comprise a HCDR1 as set forth in SEQ ID NO: 6, a HCDR2 as set forth in SEQ ID NO: 12 and a HCDR3 as set forth in SEQ ID NO: 18; and

[0351] (2) the VL 5T4 may comprise a LCDR1 as set forth in SEQ ID NO: 24, a LCDR2 as set forth in SEQ ID NO: 30 and a LCDR3 as set forth in SEQ ID NO: 36.

[0352] In further preferred embodiments of the aforementioned embodiments, the domain specifically binding to 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0353] (1) the VH 5T4may comprise an amino acid sequence as set forth in SEQ ID NO: 47, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence set forth in SEQ ID NO: 47 and wherein all amino acid differences are in non-CDR regions; and

[0354] (2) the VL 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 57, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence set forth in SEQ ID NO: 57 and wherein all amino acid differences are in non-CDR regions.

[0355] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0356] In further preferred embodiments, the domain that specifically binds 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0357] (1) the VH 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 47; and

[0358] (2) the VL 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 57.

[0359] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds 5T4 can be an antibody or antigen-binding fragment thereof that comprises a constant region. In such embodiments, the anti-5T4 antibody is preferably a humanized antibody.

[0360] In certain embodiments, the antibody or antigen-binding fragment thereof can be an scFv.

[0361] In certain embodiments, in the scFv that specifically binds to 5T4, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv that specifically binds to 5T4 can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds to 5T4, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0362] In certain embodiments, the antibody that specifically binds to 5T4 can be a monospecific antibody that specifically binds to 5T4.

[0363] In certain embodiments, the antibody that specifically binds to 5T4 can be a single chain antibody that specifically binds to 5T4. The single chain antibody can comprise any of the foregoing scFv that specifically binds to 5T4 and necessary components.

[0364] In certain embodiments, the antibody that specifically binds to 5T4 can be a conventional antibody that specifically binds to 5T4, preferably, can be an IgG class antibody. The conventional antibody can comprise any of the foregoing VH portion, VL portion, and necessary linker sequence, constant region components of the domain that specifically binds to 5T4.

[0365] In certain embodiments, the antibody that specifically binds to 5T4 can be a multispecific antibody that specifically binds to 5T4.

[0366] In certain embodiments, the multispecific antibody can be a bispecific antibody. For example, can be an anti-5T4xCD28 antibody. The anti-5T4xCD28 antibody can comprise any of the foregoing domain that specifically binds to CD28.

[0367] In certain embodiments, the multispecific antibody can be a trispecific antibody.

[0368] EGFR

[0369] In some embodiments, the TAA is EGFR, and the antibody or antigen-binding fragment thereof comprises a domain that specifically binds to EGFR. The antibody or antigen-binding fragment thereof that specifically binds to EGFR can be used for the production of a medicament for treating an EGFR-positive disease, preferably a tumor.

[0370] In certain embodiments, the domain that specifically binds to EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0371] (1) the VH EGFR may comprise a HCDR1 as set forth in SEQ ID NO: 5, a HCDR2 as set forth in SEQ ID NO: 11, and a HCDR3 as set forth in SEQ ID NO: 17; and

[0372] (2) the VL EGFR may comprise a LCDR1 as set forth in SEQ ID NO: 23, a LCDR2 as set forth in SEQ ID NO: 29, and a LCDR3 as set forth in SEQ ID NO: 35.

[0373] In further preferred embodiments of the above embodiments, the domain that specifically binds to EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0374] (1) the VH EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 46, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 46 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0375] (2) the VL EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 56, or an amino acid sequence that has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 56 and all amino acid differences are in the amino acid sequence of the non-CDR regions.

[0376] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0377] In further preferred embodiments, the domain specifically binding to EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). In which

[0378] (1) the VH EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 46; and

[0379] (2) the VL EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 56.

[0380] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binding to EGFR can be an antibody or antigen-binding fragment thereof comprising a constant region. In such embodiments, the anti-EGFR antibody is preferably a humanized antibody.

[0381] In certain embodiments, the antibody or antigen-binding fragment thereof can be an scFv.

[0382] In certain embodiments, in the scFv specifically binding to EGFR, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv specifically binding to EGFR can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n or (GGS)nGG, or (GGGGS)n, (GGS)n or (GGS)nGG, and n is an integer of 1-10, preferably an integer of 1-5, such as 1, 2, 3, 4 or 5. In specific embodiments, in the scFv specifically binding to EGFR, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215 or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0383] In certain embodiments, the antibody specifically binding to EGFR can be a monospecific antibody specifically binding to EGFR.

[0384] In certain embodiments, the antibody that specifically binds to EGFR can be a single-chain antibody that specifically binds to EGFR. The single-chain antibody can comprise the scFv that specifically binds to EGFR of any one of the foregoing and necessary components.

[0385] In certain embodiments, the antibody that specifically binds to EGFR can be a conventional antibody that specifically binds to EGFR, preferably, can be an IgG class antibody. The conventional antibody can comprise the VH portion, the VL portion of the domain that specifically binds to EGFR of any one of the foregoing and necessary linker sequence, constant region components.

[0386] In certain embodiments, the antibody that specifically binds to EGFR can be a multispecific antibody that specifically binds to EGFR.

[0387] In certain embodiments, the multispecific antibody can be a bispecific antibody. For example, can be an anti-EGFRxCD28 antibody. The anti-EGFRxCD28 antibody can comprise the domain that specifically binds to CD28 of any one of the foregoing.

[0388] In certain embodiments, the multispecific antibody can be a trispecific antibody.

[0389] TAAxCD3 bispecific antibody

[0390] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TAA and CD3 can be a bispecific antibody.

[0391] Specifically, the scFv that specifically binds to TAA can comprise, in order from N-terminus to C-terminus, a VH TAA , an intradomain linker, and a VL TAA , or can comprise, in order from N-terminus to C-terminus, a VL TAA , an intradomain linker, and a VH TAA ; and the scFv that specifically binds to CD3 can comprise, in order from N-terminus to C-terminus, a VH CD3 , an intradomain linker, and a VL CD3 , or can comprise, in order from N-terminus to C-terminus, a VL CD3 , an intradomain linker, and a VH CD3 .

[0392] Further, the bispecific antibody can comprise, in order from N-terminus to C-terminus: the scFv that specifically binds to TAA, an interdomain linker, the scFv that specifically binds to CD3; which can specifically produce the following combinations of domain connections: (1) VH TAA -intradomain linker-VL TAA -interdomain linker-VH CD3 -intradomain linker-VLCD3 ; (2) VL TAA - intra-domain linker - VH TAA - inter-domain linker - VH CD3 - intra-domain linker - VL CD3 ; (3) VH TAA - intra-domain linker - VL TAA - inter-domain linker - VL CD3 - intra-domain linker - VH CD3 ; or (4) VL TAA - intra-domain linker - VH TAA - inter-domain linker - VL CD3 - intra-domain linker - VH CD3 .

[0393] In some embodiments, the inter-domain linker can comprise an amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 216.

[0394] In some embodiments, the bispecific antibody can comprise a constant region.

[0395] TAA x CD28 bispecific antibody

[0396] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TAA and CD28 can be a bispecific antibody.

[0397] Specifically, the scFv that specifically binds to TAA can comprise, in order from N-terminus to C-terminus, VH TAA , an intra-domain linker, and VL TAA , or comprise, in order from N-terminus to C-terminus, VL TAA , an intra-domain linker, and VH TAA ; and the scFv that specifically binds to CD28 can comprise, in order from N-terminus to C-terminus, VH CD28 , an intra-domain linker, and VL CD28 , or comprise, in order from N-terminus to C-terminus, VL CD28 , an intra-domain linker, and VH CD28 .

[0398] Further, the bispecific antibody can comprise, in order from N-terminus to C-terminus, the scFv that specifically binds to TAA, an inter-domain linker, the scFv that specifically binds to CD28; which can specifically produce the following combinations of domain connections: (1) VH TAA - intra-domain linker - VL TAA - inter-domain linker - VHCD28 - an intra-domain linker - VL CD28 ; (2) VL TAA - an intra-domain linker - VH TAA - an inter-domain linker - VH CD28 - an intra-domain linker - VL CD28 ; (3) VH TAA - an intra-domain linker - VL TAA - an inter-domain linker - VL CD28 - an intra-domain linker - VH CD28 ; or (4) VL TAA - an intra-domain linker - VH TAA - an inter-domain linker - VL CD28 - an intra-domain linker - VH CD28 .

[0399] In certain embodiments, the inter-domain linker can comprise an amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 216.

[0400] In certain embodiments, the bispecific antibody can comprise a constant region.

[0401] An antibody or antigen-binding fragment thereof that specifically binds to TAA, CD3 and CD28

[0402] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to CD28 and CD3, which can comprise the domain that specifically binds to CD28 and CD3 of any one of the preceding.

[0403] In certain embodiments, the antibody or antigen-binding fragment thereof can further comprise a domain that specifically binds to a tumor associated antigen (TAA). The TAA can be a protein that is expressed by tumor cells but not normal cells, or a protein that is expressed in higher amounts by tumor cells than normal cells, preferably a cell membrane protein. In specific embodiments, the TAA can be selected from the group consisting of EGFR, Her2, Her3, Her4, NY-ESO-1, B7-H3, CD19, CD20, CD22, CD30, CD33, CD46, CD70, CEACAM5, ENPP3, PSMA (FOLH1), c-Met, folate receptor alpha, GPC3, Claudin 18.2, MUC1, MUC2, MUC4, MUC5AC, MUC6, MUC16, MUC20, NCAM, Nectin4, 5T4, IL13ra2, LIV1 (ZIP6), CD123, CD7, GD2, PSCA, P-cadherin, STEAP-1, SLC44A4, SLITRK6, TROP2, EBV16-E7, H3.3, EGFRvIII, BCMA, and MSLN. Preferably, the TAA is selected from the group consisting of 5T4, EGFR, PSMA, and MSLN.

[0404] The antibodies or antigen-binding fragments thereof that specifically bind to a TAA, human CD3, and human CD28 protected by the present application have functional advantages, including (1) higher affinity to the human CD28 target antigen; (2) enhanced binding of the TAA binding domain to its target antigen; (3) stronger ability to induce T cell activation and T cell proliferation and differentiation; and (4) more effective tumor killing and in vivo tumor inhibition. For example, the antibodies or antigen-binding fragments thereof that specifically bind to a TAA, human CD3, and human CD28 can still have a high affinity to the human CD28 antigen. In preferred embodiments, the affinity is less than 10 nM. For another example, the antibodies or antigen-binding fragments thereof that specifically bind to a TAA, human CD3, and human CD28 enhance the binding of the TAA binding domain to its target antigen. For yet another example, the antibodies or antigen-binding fragments thereof that specifically bind to a TAA, human CD3, and human CD28 mediate stronger in vivo tumor inhibition.

[0405] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise:

[0406] (1) a first binding domain capable of specifically binding to a tumor associated antigen (TAA);

[0407] (2) a second binding domain capable of specifically binding to CD3; and / or

[0408] (3) a third binding domain capable of specifically binding to CD28.

[0409] In certain embodiments, the first binding domain can be an antibody or an antigen-binding fragment thereof comprising a constant region.

[0410] In certain embodiments, the second binding domain can be an antibody or an antigen-binding fragment thereof comprising a constant region.

[0411] In certain embodiments, the third binding domain can be an antibody or an antigen-binding fragment thereof comprising a constant region.

[0412] In certain embodiments, the first binding domain can be an scFv.

[0413] In certain embodiments, the second binding domain can be an scFv.

[0414] In certain embodiments, the third binding domain can be an scFv.

[0415] The scFv sequence as the first binding domain can be derived from an anti- corresponding TAA antibody or an antibody analog, derivative known in the art, respectively. The scFv sequence as the second binding domain can be derived from an anti-CD3 antibody or an antibody analog, derivative known in the art, respectively. The scFv sequence as the third binding domain can be derived from an anti-CD28 antibody or an antibody analog, derivative known in the art, respectively. The scFv sequence as the third binding domain can also be derived from the CD28 antibody protected in the present application (i.e., the aforementioned anti-CD28 antibody). For example, each binding domain can be an scFv comprising VH and VL derived from a known corresponding antibody. The antibody can be an animal-derived antibody such as a murine-derived antibody, a chimeric antibody such as a human-murine chimeric antibody, or a human antibody or a humanized antibody.

[0416] In certain embodiments, the scFv specifically binding to CD3 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0417] (1) the VH CD3 may comprise HCDR1, HCDR2, and HCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 205; and

[0418] (2) the VL CD3 may comprise LCDR1, LCDR2, and LCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 206.

[0419] In further preferred embodiments of the above embodiments, the scFv specifically binding to CD3 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0420] (1) the VH can comprise an amino acid sequence as set forth in SEQ ID NO: 205, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 205 and all amino acid differences are in non-CDR regions; and

[0421] (2) the VL can comprise an amino acid sequence as set forth in SEQ ID NO: 206, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 206 and all amino acid differences are in non-CDR regions.

[0422] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0423] In further preferred embodiments, the scFv that specifically binds to CD3 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0424] (1) the VH CD3 can comprise an amino acid sequence as set forth in SEQ ID NO: 205; and

[0425] (2) the VL CD3 can comprise an amino acid sequence as set forth in SEQ ID NO: 206.

[0426] In certain embodiments, in the scFv that specifically binds CD3, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv that specifically binds CD3 can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer of 1-10, preferably an integer of 1-5, for example 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds CD3, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0427] In certain embodiments, the scFv that specifically binds CD28 can comprise:

[0428] (1) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), a heavy chain complementarity determining region 3 (HCDR3); and

[0429] (2) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), a light chain complementarity determining region 3 (LCDR3).

[0430] The HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 1. The HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 7. The HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 13. The LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 19. The LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 25. The LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence at least 80% identical (such as at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% identical) to the amino acid sequence set forth in SEQ ID NO: 31.

[0431] For example, the VH can comprise an HCDR1 as set forth in SEQ ID NO: 1, an HCDR2 as set forth in SEQ ID NO: 7, and an HCDR3 as set forth in SEQ ID NO: 13; and the VL can comprise an LCDR1 as set forth in SEQ ID NO: 19, an LCDR2 as set forth in SEQ ID NO: 25, and an LCDR3 as set forth in SEQ ID NO: 31. CD28 For example, the VH can comprise an HCDR1 as set forth in SEQ ID NO: 1, an HCDR2 as set forth in SEQ ID NO: 7, and an HCDR3 as set forth in SEQ ID NO: 13; and the VL can comprise an LCDR1 as set forth in SEQ ID NO: 19, an LCDR2 as set forth in SEQ ID NO: 25, and an LCDR3 as set forth in SEQ ID NO: 31. CD28may comprise an LCDR1 as set forth in SEQ ID NO: 19, an LCDR2 as set forth in SEQ ID NO: 25, and an LCDR3 as set forth in SEQ ID NO: 31.

[0432] In further preferred embodiments of the above-mentioned embodiments, the scFv specifically binding to CD28 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0433] (1) the VH CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 37 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0434] the VL CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 48, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 48 and all amino acid differences are in the amino acid sequence of the non-CDR regions; or

[0435] (2) the VH CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 38 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0436] the VL CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 49, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 49 and all amino acid differences are in the amino acid sequence of the non-CDR regions.

[0437] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0438] In further preferred embodiments, the scFv specifically binding to CD28 can comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0439] (1) the VH CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 37, and the VL CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 48; or

[0440] (2) the VH CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 38, and the VL CD28 may comprise an amino acid sequence as set forth in SEQ ID NO: 49.

[0441] In certain embodiments, in the scFv specifically binding to CD28, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv specifically binding to CD28 can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, for example 1, 2, 3, 4, or 5. In specific embodiments, the intradomain linker between the VH and VL in the scFv specifically binding to CD28 can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0442] In certain embodiments, the antibody or antigen-binding fragment thereof can further comprise a first interdomain linker between the first and second binding domains, and / or a second interdomain linker between the second and third binding domains. In specific embodiments, the first interdomain linker can comprise an amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 216. The second interdomain linker can comprise an amino acid sequence set forth in SEQ ID NO: 217, SEQ ID NO: 218, or SEQ ID NO: 219, preferably SEQ ID NO: 217.

[0443] In certain embodiments, one or more of the first, second, and third binding domains of the present application are cross-reactive with the corresponding antigen in other species. The other species can be a laboratory animal, such as a monkey, e.g., a cynomolgus monkey (Macaca fascicularis). For example, the first binding domain of the present application is cross-reactive with a monkey TAA in addition to specifically binding to a human TAA, i.e., is also capable of specifically binding to a monkey TAA. For example, the second binding domain of the present application is cross-reactive with a monkey CD3 in addition to specifically binding to a human CD3, i.e., is also capable of specifically binding to a monkey CD3. For example, the third binding domain of the present application is cross-reactive with a monkey CD28 in addition to specifically binding to a human CD28, i.e., is also capable of specifically binding to a monkey CD28. Cross-reactivity with the corresponding monkey antigen can facilitate antibody development.

[0444] PSMA

[0445] In some specific embodiments, the TAA is PSMA. The antibody or antigen-binding fragment thereof that specifically binds to PSMA, CD3, and CD28 can comprise:

[0446] (1) a first binding domain capable of specifically binding to PSMA, the first binding domain comprising a heavy chain variable region (VH PSMA ) and a light chain variable region (VL PSMA );

[0447] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0448] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VHCD28 ) and a light chain variable region (VL CD28 ).

[0449] In preferred embodiments, the scFv that specifically binds PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0450] (1) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 10, SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0451] (2) the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22 or SEQ ID NO: 224, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34.

[0452] For example, the scFv that specifically binds PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0453] (1) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 10, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0454] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0455] (2) the VH PSMA may comprise a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 221, and a HCDR3 as set forth in SEQ ID NO: 16; and

[0456] the VL PSMA may comprise a LCDR1 as set forth in SEQ ID NO: 22, a LCDR2 as set forth in SEQ ID NO: 28, and a LCDR3 as set forth in SEQ ID NO: 34;

[0457] (3) the VH PSMAmay comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 222, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0458] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 22, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0459] (4) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 223, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0460] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0461] (5) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 10, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0462] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0463] (6) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 221, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0464] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34;

[0465] (7) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 222, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0466] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34; or

[0467] (8) the VH PSMA may comprise an HCDR1 as set forth in SEQ ID NO: 4, an HCDR2 as set forth in SEQ ID NO: 223, and an HCDR3 as set forth in SEQ ID NO: 16; and

[0468] the VL PSMA may comprise an LCDR1 as set forth in SEQ ID NO: 224, an LCDR2 as set forth in SEQ ID NO: 28, and an LCDR3 as set forth in SEQ ID NO: 34.

[0469] In further preferred embodiments of the above-mentioned embodiments, the scFv specifically binding to PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0470] (1) the VH PSMA may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241, or an amino acid sequence which is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0471] (2) the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO: 243, or an amino acid sequence which is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO: 243 and all amino acid differences are in the amino acid sequence of the non-CDR regions.

[0472] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0473] In a further preferred embodiment, the scFv specifically binding PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0474] (1) the VH PSMA may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241; and

[0475] (2) the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 242 or SEQ ID NO: 243.

[0476] Preferably, the domain specifically binding PSMA can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0477] (1) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 238, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 242;

[0478] (2) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 238, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0479] (3) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 44, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0480] (4) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 44, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 243;

[0481] (5) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 239, and the VL PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 54;

[0482] (6) the VH PSMA may comprise an amino acid sequence as set forth in SEQ ID NO: 240, and the VL PSMAmay comprise an amino acid sequence as depicted in SEQ ID NO: 54;

[0483] (7) the VH PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 241, and the VL PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 54;

[0484] (8) the VH PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 239, and the VL PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 243;

[0485] (9) the VH PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 240, and the VL PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 243; or

[0486] (10) the VH PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 241, and the VL PSMA may comprise an amino acid sequence as depicted in SEQ ID NO: 243.

[0487] In certain embodiments, in the scFv that specifically binds to PSMA, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and the VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and the VL in the scFv that specifically binds to PSMA can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, for example 1, 2, 3, 4, or 5. In specific embodiments, the intradomain linker between the VH and the VL in the scFv that specifically binds to PSMA can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0488] MSLN

[0489] In some embodiments, the TAA is MSLN. The antibody or antigen-binding fragment thereof that specifically binds to MSLN, CD3 and CD28 can comprise:

[0490] (1) a first binding domain capable of specifically binding to MSLN, the first binding domain comprising a heavy chain variable region (VH MSLN ) and a light chain variable region (VL MSLN );

[0491] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and

[0492] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0493] In preferred embodiments, the scFv that specifically binds to MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0494] (1) the VH MSLN may comprise a HCDR1 as set forth in SEQ ID NO: 3, a HCDR2 as set forth in SEQ ID NO: 9, and a HCDR3 as set forth in SEQ ID NO: 15; and

[0495] (2) the VL MSLN may comprise a LCDR1 as set forth in SEQ ID NO: 21, a LCDR2 as set forth in SEQ ID NO: 27, and a LCDR3 as set forth in SEQ ID NO: 33.

[0496] In further preferred embodiments of the above embodiments, the scFv that specifically binds to MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0497] (1) the VH MSLNmay comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 40-43 or SEQ ID NO: 45, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in any one of SEQ ID NOs: 40-43 or SEQ ID NO: 45 and all amino acid differences are in amino acid sequences of non-CDR regions; and

[0498] (2) the VL MSLN may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 51-53 or SEQ ID NO: 55, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in any one of SEQ ID NOs: 51-53 or SEQ ID NO: 55 and all amino acid differences are in amino acid sequences of non-CDR regions.

[0499] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0500] In a further preferred embodiment, the scFv specifically binding to MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0501] (1) the VH MSLN may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 40-43 or SEQ ID NO: 45; and

[0502] (2) the VL MSLN may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 51-53 or SEQ ID NO: 55.

[0503] Preferably, the scFv specifically binding to MSLN can comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0504] (1) the VH MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 40, and the VL MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 51 ;

[0505] (2) the VH MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 40, and the VL MSLNmay comprise an amino acid sequence as depicted in SEQ ID NO: 52;

[0506] (3) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 40, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 53;

[0507] (4) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 41, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 51;

[0508] (5) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 41, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 52;

[0509] (6) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 41, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 53;

[0510] (7) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 42, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 51;

[0511] (8) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 42, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 52;

[0512] (9) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 42, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 53;

[0513] (10) the VH MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 43, and the VL MSLN may comprise an amino acid sequence as depicted in SEQ ID NO: 51;

[0514] (11) the VH MSLNmay comprise an amino acid sequence as set forth in SEQ ID NO: 43, the VL MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 52; or

[0515] (12) the VH MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 43, the VL MSLN may comprise an amino acid sequence as set forth in SEQ ID NO: 53.

[0516] In certain embodiments, in the scFv that specifically binds to MSLN, the VH is located at the C-terminus or N-terminus of the VL, i.e., connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and the VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and the VL in the scFv that specifically binds to MSLN can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv that specifically binds to MSLN, the intradomain linker between the VH and the VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0517] 5T4

[0518] In some specific embodiments, the TAA is 5T4. The antibody or antigen-binding fragment thereof that specifically binds to 5T4, CD3, and CD28 can comprise:

[0519] (1) a first binding domain capable of specifically binding to 5T4, the first binding domain comprising a heavy chain variable region (VH 5T4 ) and a light chain variable region (VL 5T4 );

[0520] (2) a second binding domain capable of specifically binding to CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VLCD3 ) and

[0521] (3) a third binding domain capable of specifically binding to CD28, said third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0522] In preferred embodiments, said scFv specifically binding to 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0523] (1) said VH 5T4 may comprise a HCDR1 as set forth in SEQ ID NO: 6, a HCDR2 as set forth in SEQ ID NO: 12 and a HCDR3 as set forth in SEQ ID NO: 18; and

[0524] (2) said VL 5T4 may comprise a LCDR1 as set forth in SEQ ID NO: 24, a LCDR2 as set forth in SEQ ID NO: 30 and a LCDR3 as set forth in SEQ ID NO: 36.

[0525] In further preferred embodiments of the above embodiments, said scFv specifically binding to 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0526] (1) said VH 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 47, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 47 and all amino acid differences are in amino acid sequences of non-CDR regions; and

[0527] (2) said VL 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 57, or an amino acid sequence which has at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 57 and all amino acid differences are in amino acid sequences of non-CDR regions.

[0528] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0529] In further preferred embodiments, the scFv specifically binding 5T4 can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0530] (1) the VH 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 47; and

[0531] (2) the VL 5T4 may comprise an amino acid sequence as set forth in SEQ ID NO: 57.

[0532] In certain embodiments, in the scFv specifically binding 5T4, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and VL in the scFv specifically binding 5T4 can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n, or (GGS)nGG, or (GGGGS)n, (GGS)n, or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In specific embodiments, in the scFv specifically binding 5T4, the intradomain linker between the VH and VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0533] EGFR

[0534] In some specific embodiments, the TAA is EGFR. The antibody or antigen-binding fragment thereof specifically binding EGFR, CD3 and CD28 can comprise:

[0535] (1) a first binding domain capable of specifically binding EGFR, the first binding domain comprising a heavy chain variable region (VH EGFR ) and a light chain variable region (VL EGFR );

[0536] (2) a second binding domain capable of specifically binding CD3, the second binding domain comprising a heavy chain variable region (VH CD3) and a light chain variable region (VL CD3 ) and a light chain variable region (VL

[0537] (3) a third binding domain capable of specifically binding to CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0538] In preferred embodiments, the scFv that specifically binds to EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0539] (1) the VH EGFR may comprise a HCDR1 as set forth in SEQ ID NO: 5, a HCDR2 as set forth in SEQ ID NO: 11, and a HCDR3 as set forth in SEQ ID NO: 17; and

[0540] (2) the VL EGFR may comprise a LCDR1 as set forth in SEQ ID NO: 23, a LCDR2 as set forth in SEQ ID NO: 29, and a LCDR3 as set forth in SEQ ID NO: 35.

[0541] In further preferred embodiments of the above-mentioned embodiments, the scFv that specifically binds to EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0542] (1) the VH EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 46, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 46 and all amino acid differences are in the amino acid sequence of the non-CDR regions; and

[0543] (2) the VL EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 56, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 56 and all amino acid differences are in the amino acid sequence of the non-CDR regions.

[0544] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0545] In further preferred embodiments, the scFv specifically binding EGFR can comprise a heavy chain variable region (VH) and a light chain variable region (VL). Wherein

[0546] (1) the VH EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 46; and

[0547] (2) the VL EGFR may comprise an amino acid sequence as set forth in SEQ ID NO: 56.

[0548] In certain embodiments, in the scFv specifically binding EGFR, the VH is located at the C-terminus or N-terminus of the VL, i.e. connected in the order of NH2-VL-linker sequence-VH-COOH or NH2-VH-linker sequence-VL-COOH. In certain embodiments, the intradomain linker between the VH and the VL can be a rigid linker or a flexible linker (such as a glycine-serine polymer). In certain embodiments, the intradomain linker between the VH and the VL in the scFv specifically binding EGFR can be a first linker sequence, or a second linker sequence. In preferred embodiments, the intradomain linker can be a first linker sequence. In certain embodiments, the first linker sequence can be a linker sequence comprising (GGGGS)n, (GGS)n or (GGS)nGG, or (GGGGS)n, (GGS)n or (GGS)nGG, and n is an integer from 1 to 10, preferably an integer from 1 to 5, for example 1, 2, 3, 4 or 5. In specific embodiments, in the scFv specifically binding EGFR, the intradomain linker between the VH and the VL can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215 or SEQ ID NO: 216, preferably SEQ ID NO: 213.

[0549] TAAxCD3xCD28 trispecific antibody

[0550] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binding TAA, CD3 and CD28 can be a trispecific antibody.

[0551] Specifically, the scFv of the first binding domain can comprise, in order from N-terminus to C-terminus, a VH TAA , an intradomain linker and a VL TAA , or comprise, in order from N-terminus to C-terminus, a VL TAA , an intradomain linker and a VH TAA ; the scFv of the second binding domain can comprise, in order from N-terminus to C-terminus, a VHCD3 , an intradomain linker, and VL CD3 , or can comprise, in order from N- to C-terminus, VL CD3 , an intradomain linker, and VH CD3 ; the scFv of the third binding domain can comprise, in order from N- to C-terminus, VH CD28 , an intradomain linker, and VL CD28 , or can comprise, in order from N- to C-terminus, VL CD28 , an intradomain linker, and VH CD28 .

[0552] In the above case, the scFv of the first binding domain and the scFv of the second binding domain can be connected by a first interdomain linker, which specifically can result in the following combinations of domain connections: in order of connection from N- to C-terminus, VH TAA - intradomain linker - VL TAA - first interdomain linker - VH CD3 - intradomain linker - VL CD3 , VL TAA - intradomain linker - VH TAA - first interdomain linker - VH CD3 - intradomain linker - VL CD3 , VH TAA - intradomain linker - VL TAA - first interdomain linker - VL CD3 - intradomain linker - VH CD3 , or VL TAA - intradomain linker - VH TAA - first interdomain linker - VL CD3 - intradomain linker - VH CD3 . Likewise in the above case, the scFv of the second binding domain and the scFv of the third binding domain can be connected by a second interdomain linker, which specifically can result in the following combinations of domain connections: in order of connection from N- to C-terminus, VH CD3 - intradomain linker - VL CD3 - second interdomain linker - VH CD28 - intradomain linker - VL CD28 , VL CD3 - intradomain linker - VH CD3 - second interdomain linker - VH CD28 - intradomain linker - VL CD28 , VH CD3 - intradomain linker - VL CD3 - second interdomain linker - VL CD28 - intradomain linker - VHCD28 , or VL CD3 intra-domain linker - VH CD3 second inter-domain linker - VL CD28 intra-domain linker - VH CD28 .

[0553] Further, the trispecific antibody can comprise, in order from N-terminus to C- terminus, a scFv of the first binding domain, a first inter-domain linker, a scFv of the second binding domain, a second inter-domain linker, and a scFv of the third binding domain; which can specifically produce the following combinations of domain connections: (1) VH TAA intra-domain linker - VL TAA first inter-domain linker - VH CD3 intra-domain linker - VL CD3 second inter-domain linker - VH CD28 intra-domain linker - VL CD28 ; (2) VH TAA intra-domain linker - VL TAA first inter-domain linker - VH CD3 intra-domain linker - VL CD3 second inter-domain linker - VL CD28 intra-domain linker - VH CD28 ; (3) VL TAA intra-domain linker - VH TAA first inter-domain linker - VH CD3 intra-domain linker - VL CD3 second inter-domain linker - VH CD28 intra-domain linker - VL CD28 ; (4) VL TAA intra-domain linker - VH TAA first inter-domain linker - VH CD3 intra-domain linker - VL CD3 second inter-domain linker - VL CD28 intra-domain linker - VH CD28 ; (5) VH TAA intra-domain linker - VL TAA first inter-domain linker - VL CD3 intra-domain linker - VH CD3 second inter-domain linker - VH CD28 intra-domain linker - VL CD28 ; (6) VH TAA intra-domain linker - VL TAA first inter-domain linker - VL CD3 intra-domain linker - VHCD3 - first interdomain linker - VL CD28 - intradomain linker - VH CD28 ; (7) VL TAA - intradomain linker - VH TAA - first interdomain linker - VL CD3 - intradomain linker - VH CD3 - second interdomain linker - VH CD28 - intradomain linker - VL CD28 ; or (8) VL TAA - intradomain linker - VH TAA - first interdomain linker - VL CD3 - intradomain linker - VH CD3 - second interdomain linker - VL CD28 - intradomain linker - VH CD28 .

[0554] In preferred embodiments, the trispecific antibody can comprise an amino acid sequence as set forth in any one of SEQ NO ID: 147-154, or an amino acid sequence having at least 80% homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ NO ID: 147-154 and all amino acid differences are in non-CDR regions.

[0555] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0556] Nucleic acids, vectors, cells, and methods of making

[0557] In another aspect, the present application provides an isolated nucleic acid molecule, which can encode the antibody or antigen-binding fragment thereof of any one of the preceding.

[0558] In certain embodiments, the nucleic acid molecule can comprise one or more selected from the group consisting of the nucleotide sequences set forth in SEQ NO ID: 58-146, SEQ NO ID: 176-204, and SEQ NO ID: 207-212.

[0559] In certain embodiments, the nucleic acid molecule can encode one or more of the CDRs in the domain that specifically binds CD28. Specifically, the CDRs can be selected from the group consisting of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0560] In certain embodiments, the nucleic acid molecule can comprise one or more of the nucleotide sequences selected from the group consisting of SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, and SEQ ID NO: 212.

[0561] In certain embodiments, the nucleic acid molecule can comprise a nucleotide sequence encoding a VH CD28 and / or VL CD28 .

[0562] In certain embodiments, the nucleic acid molecule encoding a VH CD28 may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.

[0563] In certain embodiments, the nucleic acid molecule encoding a VL CD28 may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110.

[0564] In certain embodiments, the nucleic acid molecule can comprise a nucleotide sequence encoding the TAAxCD3xCD28 tri-specific antibody.

[0565] In preferred embodiments, the nucleic acid molecule encoding the TAAxCD3xCD28 tri-specific antibody can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 176-183.

[0566] In certain embodiments, the nucleic acid molecule can be purified from the genome of a cell. In the examples of the present application, the cell can be a monoclonal B lymphocyte that is specific for CD28. In certain embodiments, the method of screening can comprise: (1) injecting the experimental animal with one or more doses of a preparation of CD28 antigen; (2) obtaining peripheral blood mononuclear cells (PBMCs) from the experimental animal; and (3) isolating the desired positive B cell monoclonal.

[0567] For example, the nucleic acid molecule can be isolated from the cell by a DNA extraction kit. For another example, the nucleic acid molecule can be obtained by reverse transcription after RNA extraction from the oncolytic virus, which can be mRNA (messenger RNA).

[0568] In certain embodiments, the nucleic acid molecule can be produced or synthesized by (1) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (2) recombination by cloning, (3) purification, such as by restriction enzyme digestion and gel electrophoresis fractionation, or (4) synthesis, such as by chemical synthesis.

[0569] In certain embodiments, the nucleic acid molecule can comprise a nucleic acid molecule prepared by recombinant DNA technology.

[0570] In certain embodiments, the nucleic acid molecule can be prepared by various methods known in the art. These methods can be restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides.

[0571] In another aspect, the present application provides a vector, which can comprise the nucleic acid molecule of any one of the preceding.

[0572] In certain embodiments, the vector can comprise an expression vector.

[0573] In certain embodiments, the vector can be selected from one or more of the following: a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector.

[0574] In another aspect, the present application provides a cell, which can comprise the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, and / or the vector of any one of the preceding. Specifically, the cell is obtained by transformation of the vector of any one of the preceding. After the cell is transformed by the vector of any one of the preceding, the antibody or antigen-binding fragment thereof of any one of the preceding and / or the nucleic acid molecule of any one of the preceding can be expressed in the cell and secreted out of the cell.

[0575] In certain embodiments, the cell can comprise a host cell. Specifically, it can be an antibody-producing engineered cell.

[0576] For example, the cell can be selected from one or more of the following: a HEK293 cell, an expi293 cell, and a CHO cell.

[0577] In another aspect, the present application provides a method of producing any of the foregoing antibodies or antigen-binding fragments thereof. Specifically, the method can comprise constructing an expression vector containing a genetic sequence encoding the antibody or antigen-binding fragment thereof, then transforming the expression vector into a host cell to induce expression, and isolating the antibody or antigen-binding fragment thereof from the expression product.

[0578] In certain embodiments, the method can be a molecular biology method. For example, the method comprises preparing one or more nucleotide sequences encoding any of the foregoing antibodies or antigen-binding fragments thereof, constructing the one or more encoding nucleotide sequences into one or more expression vectors, and expressing the expression vectors in appropriate cells to prepare.

[0579] As understood by one skilled in the art, different encoding nucleotide sequences can be used, or the encoding nucleotide sequences can be optimized, given the amino acid sequence of the protein, due to the existence of codon degeneracy. Methods for codon optimization of encoding sequences are known to one skilled in the art, including adjusting codons to the host's preferred codons, reducing GC content and / or reducing GC-rich regions, and improving mRNA stability, thereby improving the expression efficiency of the target nucleotide in a particular host.

[0580] Suitable production cells are known to one skilled in the art. In some embodiments, mammalian cells, such as 293T, CHO or a derivative cell line thereof, are used as production cells. In some embodiments, microbial cells, such as bacterial cells or fungal cells, are used as production cells, for example, E. coli or yeast. In some embodiments, insect cells are used as production cells, for example, Sf9. The nucleotide sequence encoding the trispecific antibody of the present application can be codon-optimized for a specific production cell line.

[0581] The produced trispecific antibody is preferably purified. Purification of the trispecific antibody can be performed by conventional methods in the field of antibody production, which can include filtration, chromatography, and the like. The filtration step can be selected from one or more of depth filtration, ultrafiltration, diafiltration, nanofiltration. The chromatography step can be selected from one or more of affinity chromatography, cation chromatography, anion chromatography, size exclusion chromatography, hydrophobic chromatography, hydroxyapatite chromatography.

[0582] Pharmaceutical compositions, kits and uses

[0583] In another aspect, the present application provides a composition, which can comprise any of the foregoing antibodies or antigen-binding fragments thereof, any of the foregoing nucleic acid molecules, any of the foregoing vectors, any of the foregoing cells, and / or optionally a pharmaceutically acceptable carrier.

[0584] In some embodiments, the composition may comprise a pharmaceutical composition. The pharmaceutical composition may comprise an antibody or antigen-binding fragment of any of the foregoing items, a nucleic acid molecule of any of the foregoing items, a carrier of any of the foregoing items, a cell of any of the foregoing items, and / or, optionally, a pharmaceutically acceptable carrier. The optionally pharmaceutically acceptable carrier may comprise saline, buffer solution, glucose, water, glycerol, ethanol, and any combination thereof. The pharmaceutical formulation should be matched to the route of administration.

[0585] In some embodiments, the composition may comprise a pharmaceutical composition. The pharmaceutical composition may comprise: any of the aforementioned antibodies that specifically bind to CD28 or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or its antigen-binding fragment, a vector comprising the nucleic acid encoding the antibody or its antigen-binding fragment, a cell comprising the antibody or its antigen-binding fragment, the nucleic acid, and / or the vector, and / or, optionally, a pharmaceutically acceptable carrier.

[0586] In some embodiments, the composition may comprise a pharmaceutical composition. The pharmaceutical composition may comprise any of the aforementioned antibodies that specifically bind to CD28 and CD3, or antigen-binding fragments thereof, nucleic acids encoding the antibody or antigen-binding fragments thereof, a vector comprising the nucleic acid encoding the antibody or antigen-binding fragments thereof, cells comprising the antibody or antigen-binding fragments thereof, the nucleic acid, and / or the vector, and / or, optionally, a pharmaceutically acceptable carrier.

[0587] In some embodiments, the composition may comprise a pharmaceutical composition. The pharmaceutical composition may comprise an antibody or antigen-binding fragment thereof that specifically binds to any of the aforementioned CD28, CD3, and TAA (e.g., PSMA, EGFR, MSLN, and / or 5T4), a nucleic acid encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid encoding the antibody or antigen-binding fragment thereof, a cell comprising the antibody or antigen-binding fragment thereof, the nucleic acid, and / or the vector, and / or, optionally, a pharmaceutically acceptable carrier.

[0588] In some embodiments, the pharmaceutical composition can be used alone to treat a disease.

[0589] In some embodiments, the pharmaceutical composition may be used in combination with other drugs to treat the disease. These other drugs may be selected from one or more of the following: small molecule targeted anticancer agents, antibody drugs, adoptive cell therapy, oncolytic viruses, and oncolytic virus enhancers.

[0590] In particular embodiments, the other drug can comprise a small molecule targeted anticancer agent. The small molecule targeted anticancer agent can comprise a tyrosine kinase inhibitor, and / or, a serine / threonine kinase inhibitor. The tyrosine kinase can be selected from one or more of the following: epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor receptor (FGFR), Janus kinase (JAK), and focal adhesion kinase (FAK). The serine / threonine kinase can be selected from one or more of the following: cyclin-dependent kinase (CDK), mitogen-activated protein kinase (MAPK), protein kinase D (PKD), DNA-dependent protein kinase (DNA-PK), Aurora protein kinase, and pancreatic kallikrein.

[0591] In particular embodiments, the other drug can comprise a small molecule targeted anticancer agent. The target of the small molecule targeted anticancer agent can be selected from one or more of the following: a cell membrane target, a cytoplasmic target, and a nuclear target. The cell membrane target can be selected from one or more of the following: EGFR, EGFR-TK, ALK, MET, KRAS, NRAS, HER-1, HER-2, and BCR-ABL. The cytoplasmic target can be selected from one or more of the following: mTOR, VEGF, BRAF, and 26S proteasome. The nuclear target can comprise a nucleotide methyltransferase and / or a histone deacetylase.

[0592] In particular embodiments, the other drug can comprise a small molecule targeted anticancer agent. The target of the small molecule targeted anticancer agent can comprise a target against angiogenesis. The target against angiogenesis can be selected from one or more of the following: TNF-a, VEGF, VEGFR, COX-2, RAF, PDGFR, FLT, c-Kit, and KIT.

[0593] In specific embodiments, the other drug can comprise an antibody drug. The target of the antibody drug can comprise an immune checkpoint, a cytokine, and / or a costimulatory molecule. The immune checkpoint can be selected from one or more of PD-1, PD-L1, PD-L2, CTLA4, VISTA, CD155, TIGIT, ICOS, GITR, GITRL, TIM-3, CD137, and LAG-3. The cytokine can be selected from one or more of IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-a, and IFN-g. The costimulatory molecule can be selected from one or more of CD137, CD27, CD2, CD7, CD8A, CD8B, CD40, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7H3, 2B4, Fc epsilon R1 gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, and MyD88.

[0594] In specific embodiments, the other drug can comprise an adoptive cell therapy. The adoptive cell therapy can comprise a therapy in which a host immune component is extracted outside of the body, modified for immune activity, and reinfused into the same host. The host immune component can comprise immune cells, which can be selected from one or more of T lymphocytes, tumor infiltrating lymphocytes (TILs), natural killer cells (NK cells), macrophages, and B cells. The modification can comprise chimeric antigen receptor (CAR) modification and / or engineered T cell receptor (TCR) modification.

[0595] For example, the adoptive cell therapy can be selected from one or more of CAR-T, CAR-NK, CAR-M, TCR-T, TCR-NK, and TCR-M.

[0596] In specific embodiments, the other drug can comprise an oncolytic virus enhancer.

[0597] In specific embodiments, the other drug can comprise an oncolytic virus. The oncolytic virus can be selected from one or more of adenovirus, reovirus, herpesvirus, poxvirus, paramyxovirus, rhabdovirus, picornavirus, influenza virus, parvovirus, and variants of these viruses.

[0598] In certain embodiments, the composition can comprise an unisolated expression product described herein. Specifically, the expression product is an intermediate product produced from the aforementioned method of producing an antibody or antigen-binding fragment thereof, which is a mixture comprising the antibody or antigen-binding fragment thereof claimed in the present application. In certain embodiments, the expression product can be a homogenous bulk solution. Preparation and analysis of the expression product can be well known in the art.

[0599] In another aspect, the present application provides a kit comprising the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding.

[0600] In certain embodiments, the kit is used for detecting the content of CD3, CD28, and / or TAA (e.g., PSMA, EGFR, 5T4, and / or MSLN) in the mixture.

[0601] In certain embodiments, the kit is used for detecting the content of CD3 and / or CD28 in the mixture. In certain embodiments, the detection can be qualitative or quantitative. In certain examples, the content of CD3 and / or CD28 can be used to characterize the immunocompetence of the mixture, the abundance of immune cells (such as T cells) in the mixture, or the risk of developing a relevant disease (such as an immunodeficiency disease, a tumor), alone or in combination with other indicators.

[0602] In certain embodiments, the kit is used for detecting the content of the corresponding TAA in the mixture. For example, the corresponding TAA can be selected from one or more of PSMA, EGFR, 5T4, and MSLN. In certain embodiments, the detection can be qualitative or quantitative. In certain examples, the content of the TAA can be used to characterize the immunocompetence of the mixture, the abundance of immune cells (such as T cells) in the mixture, or the risk of developing a relevant disease (such as an immunodeficiency disease, a tumor), alone or in combination with other indicators.

[0603] In another aspect, the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding is used, alone or in combination with other therapies, for treating a disease and / or a condition. Specifically, the antibody or antigen-binding fragment thereof of any one of the preceding, the nucleic acid molecule of any one of the preceding, the vector of any one of the preceding, the cell of any one of the preceding, and / or the pharmaceutical composition of any one of the preceding is used for the manufacture of a medicament for treating a disease.

[0604] The present application relates to a method of treating cancer in a subject in need thereof, which can include administering to the subject a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment thereof and / or a composition as described herein. As used herein, a "subject" can include any animal that exhibits symptoms of a disease, disorder, or condition that can be treated with the antibodies or antigen-binding fragments thereof, compositions, and methods disclosed herein. Suitable subjects (e.g., patients) can include non-human primates and / or human patients. Non-human primates can be selected from one or more of the following: laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses or cows), domestic animals, pets (e.g., cats or dogs).

[0605] The present application describes "administering" as referring to introducing any of the foregoing antibodies or antigen-binding fragments thereof, any of the foregoing nucleic acid molecules, any of the foregoing vectors, any of the foregoing cells, and / or any of the foregoing pharmaceutical compositions into the body of a subject, or bringing any of the foregoing antibodies or antigen-binding fragments thereof, any of the foregoing nucleic acid molecules, any of the foregoing vectors, any of the foregoing cells, and / or any of the foregoing pharmaceutical compositions into contact with a cell and / or tissue. Administration can be by injection, irrigation, inhalation, consumption, electroosmosis, hemodialysis, iontophoresis, and / or other methods known in the art. The route of administration can vary depending on the location and nature of the disease being treated. The route of administration can include the site of administration and the mode of administration. The site of administration can be selected from one or more of the following: aural, buccal, conjunctival, cutaneous, dental, intrauterine, endosinusial, intracheal, enteric, epidural, interstitial, intraarticular, intraarterial, intraabdominal, intraaural, intra biliary, intrabronchial, intrabursa, intracavernous, intracerebral, intracisternal, intracorneal, intracoronary, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intraduodenal, intradural, intrapericardial, intradermal, esophageal, gastric, gingival, hepatic, ileal, intralesional, intralingual, intraluminal, intralymphatic, intramammary, intramedullary, intramembranous, intramuscular, intranasal, intranodal, intraocular, intraretinal, intraovarian, intraperitoneal, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intraruminal, endosinusial, intraspinous, intrasynovial, intratendinous, intratesticular, intratracheal, intrathecal, intrathoracic, intracanalicular, intratumoral, intratympanic, intrauterine, intraperitoneal, intravascular, intraventricular, intravesical, intravestibular, intravenous, intravitreal, laryngeal, nasal, nasogastric, oral, ocular, oropharyngeal, parenteral, percutaneous, periarticular, epidural, perineural, periodontal, respiratory, retrobulbar, rectal, spinal, subarachnoid, subconjunctival, subcutaneous, subdermal, subgingival, sublingual, submucosal, subretinal, topical, transdermal, endomembranous, transmucosal, transplacental, transtracheal, trans tympanic, ureteral, urethral, and vaginal. The mode of administration can be selected from one or more of the following: perfusion, lavage, and direct injection.

[0606] “Treating” or“treatment” as described herein refers to the application of a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described herein and / or a composition thereof to a subject to improve the subject’s disease or condition, or symptoms of the disease or condition. The improvement can be any improvement or amelioration of the disease or condition, or symptoms of the disease or condition. The improvement can be an observable or measurable improvement, as well as an improvement in the general feeling of well-being of the subject. Thus, the skilled artisan recognizes that treatment can improve a disease state, but can not be a complete cure of the disease. A“prophylactically effective amount” refers to an amount of a virus, viral stock, or composition effective to achieve the desired prophylactic result. As used herein,“prevention” can refer to the complete prevention of a disease symptom, the delay of onset of a disease symptom, or the reduction in severity of a subsequently occurring disease symptom. Generally, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose can be used before or at an earlier stage of disease in a subject.

[0607] In certain embodiments, the disease and / or condition can comprise a tumor. The tumor can comprise a solid tumor.

[0608] For example, the solid tumor can be selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioma, head and neck squamous carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, stomach cancer, thymic carcinoma, and endometrial cancer.

[0609] In certain embodiments, the disease and / or condition can comprise a tumor. The tumor can comprise a non-solid tumor.

[0610] For example, the non-solid tumor can comprise a hematological tumor. The hematological tumor can comprise a leukemia and / or a lymphoma. For another example, the non-solid tumor can comprise a hematological tumor. The hematological tumor can be selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin’s lymphoma, myeloma, myelodysplastic syndrome, and plasmacytoma.

[0611] In certain embodiments, the disease and / or condition can comprise an immunodeficiency disease.

[0612] For example, the immunodeficiency disease can be selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, post-surgical complications, and Sjogren-Larsson syndrome.

[0613] The trispecific antibodies of the present application can be delivered by conventional methods, preferably by systemic administration, for example by intravenous infusion.

[0614] The antibodies of the present application can be administered in a therapeutically effective amount. A "therapeutically effective amount" means the amount of an antibody that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or condition, is sufficient to effect such treatment for the disease, condition, or symptom. The "therapeutically effective amount" can vary with the antibody, the disease, condition, and / or symptoms of the disease or condition, the severity of the disease, condition, and / or symptoms of the disease or condition, the age of the subject to be treated, and / or the body weight of the subject to be treated.

[0615] In a preferred embodiment of the present application, the amount of antibody administered (mg / kg) is based on the body weight of the subject. For example, a single administration dose of the trispecific antibody of the present application can range from about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably from 5 ng / kg body weight to about 2.5 mcg / kg body weight, more preferably from 0.1 mcg / kg body weight to about 1 mcg / kg body weight. For example, administration can be at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mcg / kg body weight.

[0616] One advantage of the trispecific antibodies of the present application is that they can efficiently and completely activate T cells without the risk of post-activation apoptosis, and can induce memory T cells to provide a sustained immune response, thereby allowing administration at a relatively low administration interval. In a preferred embodiment, when administered by intravenous infusion, the trispecific antibodies of the present application can have an interval of no less than 1 day, no less than 3 days, no less than 5 days, or even a week between two administrations. For example, the trispecific antibodies of the present application can be administered 7 times per week, 5 times per week, 3 times per week, twice per week, or once per week. The trispecific antibodies of the present application can be administered in a cycle of 2-4 weeks, for example, 3 weeks, and administered for one cycle or multiple cycles. The trispecific antibodies of the present application can have an advantage over other antibodies, such as TAAxCD3 bispecific antibodies, when administered at a frequency of once per week at an equivalent dose (e.g., at an equivalent molar dose).

[0617] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the antibodies or antigen-binding fragments thereof, the preparation method and use of the present application, and are not intended to limit the scope of the present application.

[0618] Examples

[0619] Example 1

[0620] Preparation and screening of rabbit recombinant monoclonal antibody specifically binding to human CD28

[0621] The present embodiment is to immunize New Zealand white rabbits with human CD28 antigen, isolate and obtain antigen-specific B lymphocytes, screen positive B lymphocytes by ELISA and FACS methods, and obtain rabbit monoclonal antibody sequence by gene cloning.

[0622] The first immunization emulsified the complete adjuvant and the antigen in equal volume, the immunization dose was 300 μg per rabbit, the subsequent immunization emulsified the incomplete adjuvant and the antigen in equal volume, the immunization dose was 150 μg per rabbit, the injection method was subcutaneous injection on the back, a total of four immunizations, and the serum titer was detected by indirect enzyme-linked immunosorbent assay (ELISA). The rabbit peripheral anticoagulant blood was collected aseptically, the peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation, the B lymphocytes were separated by human CD28 immunomagnetic beads, the separated B lymphocytes were placed into 96-well cell culture plates after limited dilution and cultured for 6 days, and the culture supernatant was collected for antibody screening by ELISA. The Jurkat cells naturally expressing human CD28 antigen were used, and the ELISA positive B lymphocyte supernatant was screened by flow cytometry (FACS) method, and the B lymphocyte monoclonal 2H1 with high FACS positive rate was obtained. The RNA of the positive antibody corresponding B lymphocytes was extracted and reversely transcribed into cDNA, and the primers were designed to amplify the antibody heavy chain or light chain variable region gene. The amplified antibody heavy and light chain variable region genes were constructed into expression vectors and transiently transfected into expi293 cells (Gibco) for expression, and the obtained antibody was subjected to FACS binding function detection, and the positive binding antibody was subjected to gene sequencing to obtain the variable region gene sequence of rabbit monoclonal antibody 2H1 as shown in SEQ ID NO: 58 and SEQ ID NO: 102, and the corresponding amino acid sequence as shown in SEQ ID NO: 37 and SEQ ID NO: 48. The CDR region amino acid sequence and DNA sequence defined according to the Kabat principle are shown in Table 1.

[0623] Table 1 Amino acid sequence and DNA sequence of rabbit monoclonal antibody 2H1

[0624] Example 2

[0625] Humanization of rabbit-derived antibody specific to human CD28

[0626] The CDR grafting method was used to humanize the rabbit-derived 2H1 antibody in this embodiment. The V region sequence of the parent antibody was queried and aligned in the database to determine the highest similarity human Germline. After defining the CDR and framework regions of the parent antibody, the back mutation was designed according to the difference sites of the framework region. The humanized hu2H1 antibody protein was obtained after gene synthesis, plasmid construction, protein expression and purification, and identification. The antigen binding activity of the humanized antibody was detected by flow cytometry. The real-time interaction of the humanized antibody with human CD28 was detected by the principle of surface plasmon resonance (SPR), and the affinity kinetic constant was calculated. The detection results are shown in Table 2, and the KD value is 2.47E -10 (M). The heavy and light chain amino acid sequences of the humanized antibody are shown in SEQ ID NO: 38 and SEQ ID NO: 49, and the DNA sequences are shown in SEQ ID NO: 59-66 and SEQ ID NO: 103-110.

[0627] Table 2 Affinity kinetic detection results of humanized 2H1 antibody (hu2H1)

[0628] Example 3

[0629] Humanization of anti-MSLN murine antibody

[0630] The CDR grafting method was used to humanize the amino acid sequence of the anti-MSLN murine antibody Amazuximab in this embodiment. The variable region sequence of the anti-MSLN murine antibody Amazuximab was derived from the patent US7081518B1, and the heavy and light chain variable region amino acid sequences are shown in SEQ ID NO: 93 and 138. First, the parent sequence was modeled using homology modeling method. The V region sequences of the heavy and light chains of the parent antibody were queried and aligned in the database to determine the highest similarity human Germline. After defining the CDR and framework regions of the parent antibody, the back mutation was designed according to the difference sites of the framework region to obtain different degrees of humanization sequences.

[0631] Based on the parent sequence, 12 humanized molecules were designed (see Table 3). The antigen binding polypeptide after humanization was obtained after gene synthesis, plasmid construction, protein expression and purification, and identification. The binding activity of different humanized antibodies to human MSLN antigen protein (Hu-MSLN-His) was evaluated by ELISA, and the results are shown in Table 4, and the affinity kinetic detection results are shown in Table 5. The results show that the binding activity or affinity of the 12 humanized molecules obtained to the human MSLN target antigen is similar to that of the parent murine antibody to the human MSLN target antigen.

[0632] The thermal stability of humanized antibodies was assessed using differential scanning fluorometry (DSF). m The value was detected using SYPRO Orange dye. SYPRO Orange is an environmentally sensitive hydrophobic dye. When the temperature rises, the protein unfolds, exposing the hydrophobic portion. The dye specifically binds to the hydrophobic portion of the protein, resulting in enhanced fluorescence. m A higher value indicates greater protein stability. T cells in humanized antibodies... m The results of the temperature detection are shown in Table 6. The results show that the Tm1 values ​​of the 12 humanized molecules obtained are between 60.03 and 68.38 °C, and the Tm2 values ​​are between 67.65 and 82.66 °C.

[0633] Table 3. Summary of Humanized Antibody Design

[0634] Table 4. Summary of EC50 results for ELISA detection of humanized antibodies binding to human MSLN antigen.

[0635] Table 5. Results of affinity kinetics assay for humanized antibodies

[0636] Table 6. Summary of Tm values ​​for humanized antibody molecules

[0637] Example 4

[0638] Humanization of Anti-PSMA Murine Antibody

[0639] In this embodiment, the amino acid sequence of the anti-PSMA murine antibody J591 was humanized using the CDR transplantation method. The sequence of the anti-PSMA murine antibody J591 is derived from patent US6,107,090, and its heavy and light chain variable region amino acid sequence is as follows:

[0640] The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:252:

[0641] The amino acid sequence of the light chain variable region as shown in SEQ ID NO:253:

[0642] First, the maternal sequence was modeled using homology modeling. The V region sequences of the maternal antibody heavy and light chains were compared and queried in a database to determine the human germline with the highest similarity. After defining the maternal antibody CDR and frame region, reversion mutations were designed based on the differential sites in the frame region to obtain sequences with different degrees of humanization.

[0643] Ten humanized molecules were designed based on the parental sequence (see Table 7). After gene synthesis, plasmid construction, protein expression, purification and identification, the humanized antibody protein was obtained. The binding activity of different humanized antibodies was evaluated by ELISA, and the results are shown in Table 8, and the affinity kinetic detection results are shown in Table 9. The results show that the binding activity or affinity of P02692, P02693, P02694, P02695, P02696 and P02699 in the obtained humanized molecules to human PSMA target antigen is similar to that of the parental mouse antibody to human PSMA target antigen.

[0644] Among them, PSM-VH2-PSM-VL2 (P02694) is used for subsequent trispecific antibody construction, which is named huJ591 or huPSMA.

[0645] Table 7. Humanized antibody design summary table

[0646] Table 8. ELISA method for detecting the binding of humanized antibodies to human PSMA antigen EC50 results summary table

[0647] Table 9. Humanized antibody molecule affinity kinetic detection results

[0648] Example 5

[0649] Design of trispecific antigen binding polypeptides of the present application

[0650] The present embodiment provides a trispecific antigen binding polypeptide (Trispecific T-cell Engager, TriTE), which comprises (1) a first binding domain capable of specifically binding to a human tumor associated antigen (TAA), the first binding domain comprising a heavy chain variable region (VH TAA ) and a light chain variable region (VL TAA ); (2) a second binding domain capable of specifically binding to human CD3, the second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and (3) a third binding domain capable of specifically binding to human CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

[0651] The first binding domain, the second binding domain and the third binding domain of the trispecific antigen-binding polypeptide described in the present application are all scFv fragments. Specifically, the scFv of the first binding domain can comprise, in order from N-terminus to C-terminus, a VH TAA , a first linker sequence and a VL TAA , or comprise, in order from N-terminus to C-terminus, a VL TAA , a first linker sequence and a VH TAA ; the scFv of the second binding domain can comprise, in order from N-terminus to C-terminus, a VH CD3 , a first linker sequence and a VL CD3 ; the scFv of the third binding domain can comprise, in order from N-terminus to C-terminus, a VH CD28 , a first linker sequence and a VL CD28 . The scFv fragment of the first binding domain is connected to the scFv fragment of the second binding domain through the first linker sequence; the scFv fragment of the second binding domain is connected to the scFv fragment of the third binding domain through the second linker sequence.

[0652] The first binding domain of the present embodiment is designed for trispecific antigen-binding polypeptide with four different tumor-associated antigens, including PSMA, MSLN, 5T4 and EGFR. Among them, the anti-MSLN antibody sequence adopts Amazuximab or the antibody sequence P110775 (AMS-VH2-AMS-VL1) of Amazuximab humanized obtained in Embodiment 3 of the present application, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 41 and SEQ ID NO: 51; the anti-PSMA antibody sequence adopts the antibody sequence huJ591 (PSM-VH2-PSM-VL2) of J591 humanized obtained in Embodiment 4 of the present application, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 44 and SEQ ID NO: 54; the anti-EGFR antibody adopts Panitumumab, the amino acid sequence of which is derived from patent US6235883 B1, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 46 and SEQ ID NO: 56; and the anti-5T4 antibody adopts huA265, the amino acid sequence of which is derived from patent CN2024102192813, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 47 and SEQ ID NO: 57.

[0653] The second binding domain anti-CD3 antibody sequence is derived from patent WO / 1999 / 054440A1, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 205 and 206. The third binding domain anti-CD28 antibody sequence is derived from the above-mentioned Examples 1 and 2, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 38 and 49. The first binding domain specifically binding to TAA is linked to the second binding domain specifically binding to CD3 and the third binding domain specifically binding to CD28, respectively, in the manner described above. Anti-TAA x anti-CD3 x anti-CD28 trispecific antigen-binding polypeptides shown in Table 10 are constructed.

[0654] The present example further constructs trispecific antigen-binding polypeptides based on anti-CD28 antibody TGN1412 as a control for comparison with the trispecific antigen-binding polypeptides constructed based on the anti-CD28 antibody sequences derived from Examples 1 and 2 described above. The TGN1412 antibody sequence is derived from patent US20090246204A1, the heavy and light chain variable region amino acid sequences of which are shown as SEQ ID NO: 39 and 50. The design of the control trispecific antigen-binding polypeptides is shown in Table 11.

[0655] Table 10: Design of anti-TAA x anti-CD3 x anti-CD28 trispecific antigen-binding polypeptides (TriTE)

[0656] Table 11: Design of control trispecific antigen-binding polypeptides Note: (1) The spatial configuration of the SAR-SEQ64, SAR-SEQ65, and SAR-SEQ61 molecules of the control group is shown in Figure 3E (Reference: Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86-98 (2020)).

[0657] Example 6

[0658] Construction, expression, and purification of eukaryotic expression vectors of the trispecific antigen-binding polypeptides of the present application

[0659] The present embodiment uses a mammalian cell expression system to express the antibody. First, the nucleotide sequence encoding the trispecific antigen binding polypeptide as described above is codon-optimized, and the optimized nucleotide sequence is synthesized by gene synthesis method. Then, the synthesized sequence is cloned into the expression vector pCDNA3.1(+) by homologous recombination. The recombinant plasmid containing the nucleotide sequence encoding the trispecific antigen binding polypeptide described in the present application is transiently transfected into the expi293 (Gibco) suspension cell line by the PEI (Polysciences) mediated method for transient expression, and the cell culture supernatant is harvested by centrifugation.

[0660] The harvested supernatant is subjected to Capto L affinity chromatography column (purchased from Cytiva) to capture the target protein. The Capto L affinity chromatography purification method is as follows: after the culture supernatant is captured by Capto L affinity filler, 0.5M sodium chloride-containing phosphate buffer, pH 7.00 and 0.1M glycine buffer, pH 5.0 are used for elution, respectively; the target protein is eluted with 0.1M glycine buffer, pH 3.0. After elution, the sample is neutralized to pH 7.0 with alkaline neutralization solution, and the protein concentration is determined by OD280 absorption method. The culture supernatant of SAR-SEQ61, SAR-SEQ65, and SAR-SEQ64 is subjected to AT Protein A Diamond affinity chromatography column (purchased from BIA) to capture the target protein. The AT Protein A Diamond affinity chromatography purification method is as follows: after the culture supernatant is captured by Protein A affinity filler, 0.5M sodium chloride-containing phosphate buffer (pH 7.00) and 0.1M glycine solution (pH 5.0) are used for elution, respectively. The target protein is eluted with 0.1M glycine solution (pH 3.0), and then immediately neutralized to pH 7.0 with 0.3M disodium hydrogen phosphate. Cation exchange chromatography column (BIA) is used for fine purification to remove the aggregates. The cation exchange chromatography (SP-HP) purification method is as follows: the sample after affinity chromatography is adjusted to pH 5.0 with 1M citric acid, and is equilibrated with 50mM sodium chloride-containing citric acid buffer, pH 5.0, and eluted with the same buffer gradient with the addition of 0.5M sodium chloride. The fine-purified components are concentrated by ultrafiltration concentration tube (Millipore), and stored at -80°C for standby.

[0661] The trispecific antigen binding polypeptide prepared by the above method has an expression amount as shown in Tables 12 and 13.

[0662] Table 12: Expression amount of trispecific antigen binding polypeptide (TriTE)

[0663] Table 13: Expression level of control trispecific antigen binding polypeptide

[0664] Example 7

[0665] Affinity detection of trispecific antigen binding polypeptide to target antigen

[0666] The binding ability of the antibody molecules of the present application to human CD28 (Novoprotein, Cat#: CX81) antigen protein was detected by BLI (Bio-Layer Interferometry) method. Specifically, the BLI detection was performed using an Octet RED 96 (Pall-ForteBio) instrument, and the antigen protein immobilized biosensor was combined with different concentrations of antibody molecules. The antibody molecules were gradient diluted at a starting concentration of 50 nM, a total of 7 gradients, and the antigen immobilized biosensor captured the antibody molecules. 0.02% PBST was used as a negative control, the binding time was 180 s, and the dissociation time was 200 s. After subtracting the negative control from the sample signal, the fitting was performed using Octect analysis software DataAnalysis HT 12.0, and the KD value was calculated through the antibody binding and dissociation curves of the concentration gradient.

[0667] The results of the binding affinity to human CD28 antigen are shown in Table 14, and the results show that the trispecific antigen binding polypeptide constructed with anti-CD28 hu2H1 antibody has an affinity to human CD28 antigen between 3.70-9.24 nM. The affinity of the monovalent anti-CD28 hu2H1 to the antigen is basically maintained, i.e., the third domain of the trispecific antigen binding polypeptide can correctly bind to the CD28 target antigen.

[0668] Table 14: Affinity kinetic results of trispecific antigen binding polypeptide to human CD28

[0669] The same method was used to detect the affinity of MSLNXCD3xCD28 trispecific antigen-binding polypeptides to human MSLN (Acro, Cat#: MSN-H522a) antigen, and the results are shown in Table 15. P110772, P110775, P110778 are different humanized sequences of Amazuximab, and their affinities to human MSLN antigen are between 0.97-2.63 nM, as shown in Table 5. When these humanized sequences are constructed into trispecific antigen-binding polypeptides based on anti-CD28 TGN1412 antibody, the affinity of the first binding domain to human MSLN antigen is between 7.03-23.5 nM, which is lower than the original affinity of the humanized antibody to the antigen, which may be related to the monovalency of the anti-MSLN antibody domain in the trispecific antigen-binding polypeptide. Unexpectedly, when the humanized antibody P11075 is constructed into a trispecific antigen-binding polypeptide based on anti-CD28 hu2H1 antibody, the affinity of the first binding domain to human MSLN antigen is between 0.21-0.41 nM, which is higher than the original affinity of the bivalent humanized antibody, and also higher than the affinity of the same humanized antibody sequence constructed into a trispecific antigen-binding polypeptide based on anti-CD28 TGN1412 antibody, which may be due to the anti-CD28 hu2H1 antibody as the third binding domain of the trispecific antigen-binding polypeptide enhancing the binding of the first binding domain to its corresponding target antigen.

[0670] Similarly, when anti-EGFR antibody is constructed into a trispecific antigen-binding polypeptide based on anti-CD28 hu2H1 antibody, the affinity of the first binding domain to human EGFR antigen is 0.474 nM, which is higher than the affinity of the same anti-EGFR antibody sequence constructed into a trispecific antigen-binding polypeptide based on anti-CD28 TGN1412 antibody (0.744 nM). Or when anti-PSMA antibody is constructed into a trispecific antigen-binding polypeptide based on anti-CD28 hu2H1 antibody, the affinity of the first binding domain to human PSMA antigen is 0.99 nM, which is higher than the affinity of the same anti-PSMA antibody sequence constructed into a trispecific antigen-binding polypeptide based on anti-CD28 TGN1412 antibody (3.25 nM). Similarly, it also shows that the anti-CD28 hu2H1 antibody as the third binding domain of the trispecific antigen-binding polypeptide enhances the binding of the first binding domain to its corresponding target antigen.

[0671] Table 15: Affinity kinetics results of TAAxCD3xCD28 trispecific antigen-binding polypeptides to tumor-associated antigens TAA

[0672] Example 8

[0673] Jurkat-NFAT system in vitro to assess the T cell activation effect mediated by the trispecific antigen binding polypeptide of the present application

[0674] This example uses Jurkat-NFAT reporter cells (Novoprotein) as an evaluation system to test the ability of different specific antigen binding polypeptides shown in Table 16 to mediate the activation of effector cells. Jurkat-NFAT reporter cells are a stable Jurkat cell line expressing a firefly luciferase reporter gene. Specifically, different concentrations of the above-mentioned antigen binding polypeptides to be tested are incubated with Jurkat-NFAT and target cells human prostate cancer cells LNCaP, human papillary ovarian adenocarcinoma cells CAOV3, human lung adenocarcinoma cells DV90, human breast cancer cells HCC1937, human pancreatic cancer cells HPAC, human mesothelioma cells NCI-H2452, human epidermoid carcinoma cells A431, human colon cancer cells HT29, and then the expression of luciferase driven by the NFAT response element (NFAT-RE) after activation of Jurkat-NTAT cells is detected. By adding luciferase substrate detection and quantifying bioluminescence signal, the activation effect of the antigen binding polypeptide to be tested on T cells is observed.

[0675] Table 16: Amino acid and nucleotide sequences of trispecific antigen binding polypeptides

[0676] The specific procedure includes: (1) Take the target cells, prepare a cell suspension of 1 x 10 5 cells / mL in RPMI 1640 + 0.5% BSA, and inoculate 100 μL per well in a 96-well cell culture plate, and incubate at 37°C, 5% CO2 for 24 h to adhere; (2) Take the stable strain of Jurkat-NFAT cells (Novoprotein), prepare a cell suspension of 2.5 x 10 5 cells / mL or 6.25 x 10 5 cells / mL in RPMI 1640 + 0.5% BSA, and inoculate 80 μL per well in a 96-well cell culture plate; (3) Add gradient-diluted test sample solution, 20 μL per well; (4) Mix and incubate at 37°C, 5% CO2 for 6 h; (5) Add 30 ul KeyTecUltra Luciferase Detection reagent (VEKY-BIO) per well, mix, and read the fluorescence value RLU of each well using a microplate reader.

[0677] Results are shown in Figures 4A-4E. Under the same target cell conditions, the activation of Jurkat cells by the trispecific antigen-binding polypeptides constructed with anti-CD28 hu2H1 antibodies was superior to the activation of Jurkat cells by the trispecific antigen-binding polypeptides constructed with anti-CD28 TGN1412 antibodies.

[0678] Example 9

[0679] In vitro assessment of the killing activity of human PBMC mediated by the trispecific antigen-binding polypeptides of the present application against target cells

[0680] This example assessed the killing activity of human PBMC mediated by the different specific antigen-binding polypeptides constructed (as listed in Example 8) against target cells. Human PBMC isolated from healthy volunteers were used as effector cells, and human prostate cancer cells 22RV1, human ovarian adenocarcinoma cells CAOV3, human lung adenocarcinoma cells DV90, human breast cancer cells HCC1937, human pancreatic acinar epithelial carcinoma HPAC, human pancreatic adenocarcinoma cells BxPC-3, human mesothelioma cells NCI-H2452, human epidermal carcinoma cells A431, human colon cancer cells HT29 and HCT116 were used as target cells. After co-incubation of different concentrations of test samples with PBMC and target cells, the number of target cells in the system was detected, thereby assessing the killing activity of human PBMC mediated by TAAxCD3xCD28 trispecific antigen-binding polypeptides against different target cells.

[0681] Specifically, PBMC of healthy volunteers were extracted with Ficoll-Paque Plus (GE, Cat#: 17-1440-02) as effector cells, and the effector cells and target cells were added to 96-well flat-bottom cell culture plates (PBMC: 2x10 5 4 The test samples were serially diluted with RPIM 1640 complete medium, and then added to the above 96-well flat-bottom cell culture plates. After co-incubation at 37°C, 5% CO2 for 24h, 72h or 120h, the plates were washed and 100μL of RPIM 1640 complete medium containing 10% CCK8 stock solution was added. After further reaction at 37°C for 1-4h, the OD450 was measured by a microplate reader, and the cell killing efficiency was calculated according to the following formula; two replicates were set for each group, and the vehicle control with the addition of RPMI 1640 complete medium was used as a blank control group. The killing efficiency was calculated according to the following formula:

[0682] The data were analyzed by GraphPad Prism software, and the killing activity of human PBMC mediated by the antibodies against target cells was calculated as the 50% maximal effective concentration EC 50 ​Values represent.

[0683] Results are shown in Figures 5A-5D. Compared to trispecific antigen-binding polypeptides constructed with anti-CD28 TGN1412 antibodies, trispecific antigen-binding polypeptides constructed with anti-CD28 2H1 antibodies mediated stronger killing activity of effector cells against each tumor cell line.

[0684] Example 10

[0685] Effects of trispecific antigen-binding polypeptides of the present application on patient-derived tumor organoids

[0686] Organoids are a kind of 3D cell culture that uses the self-assembly characteristics of stem cells to culture in vitro. They have highly similar histology, genetics, and physiological characteristics to real tissues and can be used as a "miniature double" of real tissue organs. In this embodiment, the organoid model (10-2-T, provided by Shanghai Huawo New Life Science Co., Ltd.) constructed from tumor tissue derived from a patient with ovarian cancer was used to evaluate the pharmacodynamic effects of MSLNxCD3xCD28 trispecific antibodies. The specific antigen-binding polypeptides of each group are shown in Table 17.

[0687] Table 17: Amino acid and nucleotide sequences of MSLNxCD3xCD28 trispecific antigen-binding polypeptides

[0688] The specific procedure includes: (1) First, add 1000 cells / well of organoids to a 384-well plate; (2) After the organoids have settled stably at the bottom of the plate, add 20,000 cells / well of T cells. The effector-to-target ratio is 20:1; (3) After the T cells have settled uniformly at the bottom of the plate, add different concentrations of antigen-binding polypeptides in gradient dilution, and adjust the volume of each well to 100 μL, with double replicates for each group; (4) After 120 h of co-incubation at 37°C, 5% CO2, terminate the experiment, take photos of the co-incubated cells under bright field, and centrifuge to collect the supernatant for detection of lactate dehydrogenase (LDH) release. LDH is a stable cytoplasmic enzyme widely present in various organisms. Under normal circumstances, it cannot pass through the cell membrane, but when the cell is damaged or dies, it can be released outside the cell. By detecting LDH, the killing effect of antigen-binding polypeptides on target tumor cells can be reflected.

[0689] Results are shown in Figures 6A-6E. The trispecific antigen binding polypeptide SEQ65-2-1 of MSLNxCD3xCD28 had a significant effect on inducing proliferation of T cells compared to the control group (10-2-T ovarian cancer organoids + T cells); the killing effect of the trispecific antigen binding polypeptide 65-2-1 constructed with the anti-CD28 2H1 antibody was superior to the trispecific antigen binding polypeptides 65-1, 65-2, or SAR-SEQ65 constructed with the anti-CD28 TGN1412 antibody on target cells, as seen from the end-point supernatant LDH data (Figure 7).

[0690] Example 11

[0691] Tumor inhibition effect of the trispecific antigen binding polypeptide of the present application in a subcutaneously transplanted tumor-bearing model

[0692] NPG mouse subcutaneously transplanted human breast cancer HCC1954 tumor-bearing model:

[0693] This example evaluated the tumor inhibition effect of the MSLNxCD3xCD28 trispecific antigen binding polypeptide in a NPG mouse subcutaneously transplanted human breast cancer HCC1954 tumor-bearing model. Specifically, 5-6-week-old female immunodeficient NPG mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously inoculated with 5x10 6 HCC1954 cells and 2.5x10 6 PBMCs on the right side, with a volume of 200 μL. The animal operation program was approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Medical Laboratory Animal of the Chinese Academy of Medical Sciences.

[0694] When the tumor grew to about 75 mm 2 , 20 tumor-bearing mice were randomly divided into 4 groups (5 mice / group) according to body weight and tumor volume, including a solvent (PBS) control group (G1), 1 mg / kg SEQ70-2 (G2), 1 mg / kg SEQ65-2 (G3), and 2 mg / kg SAR-SEQ65 (G4). The mice in each group were intravenously administered twice a week for 4 weeks, for a total of 8 administrations. The body weight and tumor volume of the mice were measured and recorded twice a week, and the following indexes were calculated: tumor volume (V), relative tumor volume (RTV), tumor volume growth inhibition rate (TGI TV ), and tumor weight inhibition rate (IR TW %).

[0695] V = (length x width 2 ) / 2

[0696] RTV = V tV0, Vt is the tumor volume measured at time point t, and V0 is the tumor volume at the beginning of treatment.

[0697] TGI TV = (1 - T / C) x 100%, wherein T and C are the RTV of the test group and the vehicle control group at a certain time point, respectively.

[0698] IR TW %= (average tumor weight of the vehicle control group - average tumor weight of the test group) / average tumor weight of the vehicle control group x 100%.

[0699] The results are shown in Figures 8A-8C and Table 18: MSLNxCD3xCD28 trispecific antigen binding polypeptide SEQ70-2 at 1 mg / kg dose had a significant inhibitory effect on tumor growth (P < 0.01) compared to the vehicle (PBS) control (G1), and was superior to the control MSLNxCD3xCD28 trispecific antigen binding polypeptide SEQ65-2 and SAR-SEQ65. At the end of the study, tumors were excised, weighed and the tumor weight inhibition rate (IR TW %) was calculated. The results showed that the IR TW % values of G2-G4 groups at the end of the study were 73.68%, 34.60% and 7.65%, respectively. Similarly, the trispecific antigen binding polypeptide SEQ70-2 was also significantly superior to the control MSLNxCD3xCD28 trispecific antigen binding polypeptide SEQ65-2 and SAR-SEQ65 in inhibiting tumor weight.

[0700] Table 18: Tumor growth inhibition rate (TGI TV (%) over time

[0701] Although the specific embodiments of the present application are described above, it should be understood by those skilled in the art that these are merely exemplary and that various modifications or changes can be made to these embodiments without departing from the principles and spirit of the present application. Therefore, the scope of protection of the present application is defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human CD28, the antibody or antigen-binding fragment thereof comprising: (1) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 7, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 13; and (2) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 19, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 25, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO:

31.

2. The antibody or antigen-binding fragment thereof of claim 1, the VH comprising an amino acid sequence as set forth in SEQ ID NO: 37 or SEQ ID NO: 38, or comprising an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 37 or SEQ ID NO:

38.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, the VL comprising an amino acid sequence as set forth in SEQ ID NO: 48 or SEQ ID NO: 49, or comprising an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 48 or SEQ ID NO:

49.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which is selected from the group consisting of a mono-specific antibody, a multi-specific antibody, a human antibody, a humanized antibody, a chimeric antibody, a Fab, a Fv, a F(ab’)2, and a single chain Fv (scFv).

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a scFv that specifically binds to human CD28.

6. The antibody or antigen-binding fragment thereof of claim 5, the scFv that specifically binds to human CD28 comprising the VH and the VL connected by an intra-domain linker.

7. The antibody or antigen-binding fragment thereof of claim 6, the intra-domain linker is selected from a first linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO:

216.

8. The antibody or antigen-binding fragment thereof of claim 7, the intra-domain linker is selected from a first linker sequence comprising an amino acid sequence as set forth in SEQ ID NO:

213.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, which is a mono-specific antibody.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-8, which is a multi-specific antibody.

11. The antibody or antigen-binding fragment thereof of claim 10, the multispecific antibody comprising a domain that specifically binds to a tumor-associated antigen (TAA).

12. The antibody or antigen-binding fragment thereof of claim 11, the TAA is selected from the group consisting of EGFR, Her2, Her3, Her4, NY-ESO-1, B7-H3, CD19, CD20, CD22, CD30, CD33, CD46, CD70, CEACAM5, ENPP3, PSMA (FOLH1), c-Met, folate receptor alpha, GPC3, Claudin 18.2, MUC1, MUC2, MUC4, MUC5AC, MUC6, MUC16, MUC20, NCAM, Nectin4, 5T4, IL13ra2, LIV1 (ZIP6), CD123, CD7, GD2, PSCA, P-cadherin, STEAP-1, SLC44A4, SLITRK6, TROP2, EBV16-E7, H3.3, EGFRvIII, BCMA, and MSLN.

13. The antibody or antigen-binding fragment thereof of any one of claims 10-12, the multispecific antibody comprising a domain that specifically binds to a T cell surface molecule other than human CD28.

14. The antibody or antigen-binding fragment thereof of claim 13, the T cell surface molecule other than human CD28 is selected from the group consisting of CD2, CD3, CD4, CD8, CD40, CD137, OX40, and GITR.

15. The antibody or antigen-binding fragment thereof of claim 13 or 14, the T cell surface molecule other than human CD28 is CD3.

16. The antibody or antigen-binding fragment thereof of any one of claims 10-15, which is a bispecific antibody.

17. The antibody or antigen-binding fragment thereof of any one of claims 10-15, which is a trispecific antibody.

18. The antibody or antigen-binding fragment thereof of claim 17, the trispecific antibody comprising: (1) a first binding domain capable of specifically binding to a tumor associated antigen (TAA), the first binding domain comprising a heavy chain variable region (VH TAA ) and a light chain variable region (VL TAA ) (2) a second binding domain capable of specifically binding to CD3, said second binding domain comprising a heavy chain variable region (VH CD3 ) and a light chain variable region (VL CD3 ); and (3) a third binding domain capable of specifically binding CD28, the third binding domain comprising a heavy chain variable region (VH CD28 ) and a light chain variable region (VL CD28 ).

19. The antibody or antigen-binding fragment thereof of any one of claims 15-18, the domain that specifically binds to CD3 comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO: 205, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 205, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 206, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO:

206.

20. The antibody or antigen-binding fragment thereof of any one of claims 15-19, the domain that specifically binds to CD3 is a scFv that specifically binds to CD3.

21. The antibody or antigen-binding fragment thereof of claim 20, wherein the scFv that specifically binds CD3 comprises the VH and VL connected by an intra-domain linker.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the intra-domain linker is selected from a first linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO:

216.

23. The antibody or antigen-binding fragment thereof of claim 21 or 22, wherein the intra-domain linker is selected from a first linker sequence comprising the amino acid sequence of SEQ ID NO:

213.

24. The antibody or antigen-binding fragment thereof of any one of claims 20-23, wherein the scFv that specifically binds CD3 is connected to the scFv that specifically binds CD28 by an inter-domain linker.

25. The antibody or antigen-binding fragment thereof of claim 24, wherein the inter-domain linker is selected from a second linker sequence comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO:

219.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the inter-domain linker is selected from a second linker sequence comprising the amino acid sequence of SEQ ID NO:

217.

27. The antibody or antigen-binding fragment thereof of any one of claims 20-26, wherein the scFv that specifically binds CD3 is located at the N-terminus or C-terminus of the scFv that specifically binds CD28.

28. The antibody or antigen-binding fragment thereof of any one of claims 18-27, wherein the TAA is selected from the group consisting of PSMA, MSLN, 5T4, and EGFR.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the TAA is PSMA, and the first binding domain comprises: (1) a heavy chain variable region (VH PSMA ) comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 4, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 10, SEQ ID NO: 221, SEQ ID NO: 222, or SEQ ID NO: 223, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 16; and (2) a light chain variable region (VL PSMA ), comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO:22 or SEQ ID NO:224, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO:28, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO:

34.

30. The antibody or antigen-binding fragment thereof of claim 29, the VH PSMA comprises an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241, or an amino acid sequence that has at least 90% sequence homology to an amino acid sequence as set forth in any one of SEQ ID NO: 44 or SEQ ID NO: 238-241.

31. The antibody or antigen-binding fragment thereof of claim 29 or 30, the VL PSMA comprises an amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO: 243, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 54, SEQ ID NO: 242, or SEQ ID NO:

243.

32. The antibody or antigen-binding fragment thereof of claim 28, wherein the TAA is MSLN, and the first binding domain comprises: (1) a heavy chain variable region (VH MSLN ) comprising a heavy chain complementarity determining region 1 (HCDR1) as shown in SEQ ID NO: 3, a heavy chain complementarity determining region 2 (HCDR2) as shown in SEQ ID NO: 9, and a heavy chain complementarity determining region 3 (HCDR3) as shown in SEQ ID NO: 15; and (2) a light chain variable region (VL MSLN ), comprising a light chain complementarity determining region 1 (LCDR1) as shown in SEQ ID NO: 21, a light chain complementarity determining region 2 (LCDR2) as shown in SEQ ID NO: 27, and a light chain complementarity determining region 3 (LCDR3) as shown in SEQ ID NO:

33.

33. The antibody or antigen-binding fragment thereof of claim 32, the VH MSLN comprises an amino acid sequence as set forth in SEQ ID NO:45, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, or an amino acid sequence comprising at least 90% sequence homology to an amino acid sequence as set forth in SEQ ID NO:45, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:

43.

34. The antibody or antigen-binding fragment thereof of claim 32 or 33, the VL MSLN comprises an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 55, or an amino acid sequence having at least 90% sequence homology to an amino acid sequence as set forth in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO:

55.

35. The antibody or antigen-binding fragment thereof of claim 28, wherein the TAA is 5T4, and the first binding domain comprises: (1) a heavy chain variable region (VH 5T4 ), comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 6, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 12, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 18; and (2) a light chain variable region (VL 5T4 ), comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO:24, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO:30, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO:

36.

36. The antibody or antigen-binding fragment thereof of claim 35, the VH 5T4 comprises an amino acid sequence as set forth in SEQ ID NO: 47, or comprises an amino acid sequence with at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO:

47.

37. The antibody or antigen-binding fragment thereof of claim 35 or 36, wherein the VL 5T4 comprises an amino acid sequence as set forth in SEQ ID NO: 57, or comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO:

57.

38. The antibody or antigen-binding fragment thereof of claim 28, wherein the TAA is EGFR, and the first binding domain comprises: (1) a heavy chain variable region (VH EGFR ), comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 5, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 11, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 17; and (2) a light chain variable region (VL EGFR ), comprising a light chain complementarity determining region 1 (LCDR1) as shown in SEQ ID NO: 23, a light chain complementarity determining region 2 (LCDR2) as shown in SEQ ID NO: 29, and a light chain complementarity determining region 3 (LCDR3) as shown in SEQ ID NO:

35.

39. The antibody or antigen-binding fragment thereof of claim 38, the VH EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 46, or comprises an amino acid sequence with at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO:

46.

40. The antibody or antigen-binding fragment thereof of claim 38 or 39, the VL EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 56, or comprises an amino acid sequence with at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO:

56.

41. The antibody or antigen-binding fragment thereof of any one of claims 18-40, wherein the first binding domain is a scFv.

42. The antibody or antigen-binding fragment thereof of any one of claims 41, the first binding domain comprising a VH TAA and a VL TAA linked by an intradomain linker.

43. The antibody or antigen-binding fragment thereof of claim 42, wherein the intra- domain linker is selected from a first linker sequence comprising an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO:

216.

44. The antibody or antigen-binding fragment thereof of claim 43, wherein the intra- domain linker is selected from a first linker sequence comprising an amino acid sequence of SEQ ID NO:

213.

45. The antibody or antigen-binding fragment thereof of any one of claims 18-44, wherein the first binding domain is connected to the second binding domain by a first inter- domain linker.

46. The antibody or antigen-binding fragment thereof of claim 45, wherein the first inter- domain linker is selected from a first linker sequence comprising an amino acid sequence selected from any one of the group consisting of SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO:

216.

47. The antibody or antigen-binding fragment thereof of claim 46, wherein the first inter- domain linker is selected from a first linker sequence comprising an amino acid sequence of SEQ ID NO:

216.

48. The antibody or antigen-binding fragment thereof of any one of claims 18-47, wherein the first binding domain is located N-terminal to the second binding domain.

49. The antibody or antigen-binding fragment thereof of any one of claims 18-48, wherein the second binding domain is located N-terminal to the third binding domain.

50. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-49.

51. A vector comprising the nucleic acid molecule of claim 50.

52. A cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1-49, the nucleic acid molecule of claim 50, and / or the vector of claim 51.

53. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-49, the method comprising: (1) constructing an expression vector containing a gene sequence encoding the antibody or antigen-binding fragment thereof, (2) transforming the expression vector into a host cell to induce expression, and (3) isolating the antibody or antigen-binding fragment thereof from the expression product.

54. A kit for detecting CD28 content in a mixed system, comprising the antibody or antigen-binding fragment thereof of any one of claims 1-49, the nucleic acid of claim 50, the vector of claim 51, and / or the cell of claim 52.

55. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-49, the nucleic acid of claim 50, the vector of claim 51, and / or the cell of claim 52 for the manufacture of a medicament for the treatment of an immunodeficiency disease.

56. The use according to claim 55, wherein the immunodeficiency disease is selected from one or more of the group consisting of rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, post-surgical complications, and Sjogren-Larsson syndrome.

57. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-49, the nucleic acid of claim 50, the vector of claim 51, and / or the cell of claim 52 for the manufacture of a medicament for the treatment of a tumor.

58. The use according to claim 57, wherein the tumor comprises a hematological tumor and / or a solid tumor.

59. The use according to claim 58, wherein the hematological tumor is selected from one or more of the group consisting of B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, myelodysplastic syndrome, and plasmacytoma.

60. The use according to claim 58, wherein the solid tumor comprises one or more selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioma, head and neck squamous carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, stomach cancer, thymic carcinoma, and endometrial cancer.

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