Co-activated CD3 + T cell adapter antibodies and uses thereof

Abstract

The invention relates to a multispecific antigen binding protein. The antigen binding protein comprises a first binding domain capable of specifically binding MUC1, a second binding domain capable of specifically binding CD3 and a third binding domain capable of specifically binding CD28. The invention also relates to the use of the antigen binding protein for treating tumors or cancers with positive expression of MUC1.

Description

Technical Field

[0001] This application relates to the field of biomedicine and specifically to a co-activated CD3 + T cell engager antibodies and their polynucleotides, carrier molecules, pharmaceutical compositions, host cells, and uses for treating tumors or cancers. Background Art

[0002] CD3 targeting tumor-associated antigens + T cell engager (TCEs) antibodies are an emerging and promising immunotherapy. Their main mechanism of action is to form an artificial immune contact by closely linking cytotoxic T cells with tumor cells, thereby + The cytotoxic activity of T cells is redirected towards tumor cells, ultimately leading to the selective attack and lysis of targeted tumor cells. Since the FDA approved the anti-CD3 × anti-CD19 bispecific antibody Blinatumomab for the treatment of relapsed / refractory CD19-positive B-cell precursor acute lymphoblastic leukemia at the end of 2014, TCEs targeting hematological malignancies and solid tumors have been developed and demonstrated strong therapeutic effects in clinical practice, especially for hematological malignancies. However, in terms of solid tumors, there are still many challenges due to various factors such as tumor heterogeneity, immune microenvironment (TME), and tissue permeability, including: (1) the immunosuppressive characteristics of the tumor immune microenvironment (TME). Immunosuppressive cells in the tumor microenvironment include CAFs (tumor-associated fibroblasts), MDSCs (myeloid-derived suppressor cells), and Treg cells, which hinder the metabolism and activity of T cells by secreting TGF-β, IL-10, IDO, and arginase; (2) the expression of inhibitory immune checkpoint molecules. Effector T cells in the tumor immune microenvironment are in an "exhausted" state due to long-term chronic stimulation by antigens, and express immunosuppressive biomarkers such as PD-1 and CTLA-4, which inhibit anti-tumor immune activity; in addition, CD3 +T cell adaptor-mediated CD3 single signal activation will induce T cell activation death (AICD). The above factors lead to the inability of tissue-infiltrating T cells to proliferate and secrete related cytokines, thereby losing their anti-tumor ability; (3) Tumor heterogeneity. The difference in gene expression between different patients with the same malignant tumor will lead to different sensitivity of different individuals to the same TCE antibody drug. (4) Low specificity and selectivity of tumor-associated antigens. Selecting antigen targets that recognize tumor cell-specific antigens but not those on normal tissue cells is another challenge in the development of TCEs. Low specificity and selectivity of tumor antigens will enable TCEs to recognize tumor cell antigens while also recognizing antigens on normal tissue cells to kill normal cells, resulting in targeted toxicity outside tumor cells. (5) TCEs may cause the systemic rapid release of pro-inflammatory factors including IL-6, IL-10, TNFα and IFN-γ. The increase in the levels of these core cytokines will induce immune cells to produce excessive cytokines, causing a "cytokine storm", thereby affecting the dosage, limiting the efficacy of the drug, and narrowing the drug treatment window;

[0003] To address the technical limitations of T cell engager technology, the applicant proposed in application 202210822959.8 that CD28 costimulation combined with BiTEs (bispecific T-cell engagers) technology could prolong T cell survival through both exogenous and endogenous mechanisms. CD28 is one of the most important T cell co-activators discovered to date. Its exogenous mechanism primarily promotes the release of cytokines, particularly IL-2, which plays a key role in T cell growth. Its endogenous mechanism is manifested by enhancing the expression of the anti-apoptotic protein Bcl-XL (Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, Thompson CB. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity. 2010;3:87-98). In the presence of only CD3 or CD28 signals, Bcl-XL expression is weak, while in the presence of both signals, Bcl-XL expression is significantly enhanced (Watts TH. Staying alive: T cell costimulation, CD28, and Bcl-xL. J Immunol. 2010; 185: 3785-3787). In addition, CD28 co-stimulatory signals can promote the proliferation of effector T cells and memory T cells, wherein memory T cells can be rapidly activated when the body encounters the same tumor cells again, thereby effectively eliminating tumor cells. However, the above technical solution is only for hematological tumors. In the face of solid tumors with higher tumor heterogeneity (such as MUC1-positive tumors), whether this technology can overcome the above technical difficulties and simultaneously achieve specific targeting of tumor-associated antigens (TAAs) and dual-signal activation of T cells, this field still needs to provide a large number of confirmatory research results.

[0004] MUC1 is a member of the human mucin family and a transmembrane high-molecular-weight glycoprotein with a highly glycosylated extracellular domain. In normal tissues, MUC1 is expressed at the apical region of all epithelial cells, contributing to the formation of the mucus barrier and providing lubrication and anti-infection properties. In cancer cells, it has intracellular signaling functions that regulate various aspects of cancer, including cell growth, proliferation, metastasis, apoptosis, and developmental processes. MUC1 is often overexpressed in various epithelial adenocarcinomas, such as lung, liver, colon, breast, pancreatic, and ovarian cancers, and is an important tumor marker.

[0005] MUC1 is the most characteristic transmembrane mucin, and its N-terminal extracellular region includes a tandem repeat (VNTR) domain, a SEA (sea urchin sperm protein-enterokinase-aggregin) domain, and / or an EGF (epidermal growth factor)-like domain. However, in cancer-related MUC1, due to the loss of apical polar distribution of MUC1 in epithelial cells and its overexpression, its structure has also changed. The glycosylation sites in its protein core are the same as those of normal MUC1, but its glycosylation is incomplete, and the carbohydrate side chains are much shorter, exposing the core peptide that is normally hidden. The difference in low glycosylation of MUC1 on tumor cells causes a decrease in tumor cell adhesion, providing a basis for tumor metastasis. However, the overexpression of MUC1 on the surface of cancer cells and its low glycosylation characteristics also provide epitopes unique to tumor cell MUC1, providing the possibility of treatment. This application hopes to apply the principle of dual-signal activation of T cells to the design of multifunctional antibodies against mucins, especially MUC1, based on BiTE, to provide an optimized therapeutic anti-MUC1 antibody construct. Summary of the Invention

[0006] In order to overcome the defects of existing antibody technology, this application provides a co-activating CD3 that can simultaneously bind to MUC1, CD3 and CD28. + T cell engager antibodies not only retain the effectiveness and safety of multi-specific antigen binding proteins designed with existing technologies, but also creatively solve the technical problem of T cell activation-induced cell death (AICD).

[0007] On the one hand, the present application provides a co-activation CD3 + The T cell engager antibody comprises (1) a first binding domain capable of specifically binding to human tumor-associated mucin-1 (TA-MUC1), wherein the first binding domain comprises a heavy chain variable region (VH MUC1 ) and light chain variable region (VL MUC1 ); (2) a second binding domain capable of specifically binding to a human CD3 polypeptide, wherein the second binding domain comprises a heavy chain variable region (VH CD3 ) and light chain variable region (VL CD3 ); and (3) a third binding domain capable of specifically binding to a human CD28 polypeptide, wherein the third binding domain comprises a heavy chain variable region (VH CD28 ) and light chain variable region (VL CD28 ).

[0008] In some embodiments, the first binding domain, the second binding domain, and the third binding domain are single-chain antibody (scFv) fragments.

[0009] In some embodiments, the first binding domain VH MUC1 The C-terminus of the first binding domain VL MUC1 The N-terminus of the first binding domain VL is connected via a first linker sequence, or the first binding domain VL MUC1 The C-terminus of the first binding domain VH MUC1 The N-terminus of the peptide is connected via a first linker sequence.

[0010] In some embodiments, the VH of the second binding domain CD3 The C-terminus of the second binding domain VL CD3 The N-terminus of the second binding domain is connected via a first linker sequence, or the VL CD3 The C-terminus of the second binding domain VH CD3 The N-terminus of the peptide is connected via a first linker sequence.

[0011] In some embodiments, the VH of the third binding domain CD28 The C-terminus of the third binding domain VL CD28 The N-terminus of the third binding domain is connected via a first linker sequence, or the VL CD28 The C-terminus of the third binding domain VH CD28 The N-terminus of the peptide is connected via a first linker sequence.

[0012] In some embodiments, the scFv fragment of the first binding domain and the scFv fragment of the second binding domain are connected by a first linker sequence.

[0013] In some embodiments, the scFv fragment of the second binding domain and the scFv fragment of the third binding domain are connected via a second linker sequence.

[0014] In some embodiments, the antigen binding protein comprises, in order from N-terminus to C-terminus: a scFv of a first binding domain, a first linker sequence, a scFv of a second binding domain, a second linker sequence, and a scFv of a third binding domain.

[0015] In some embodiments, the connection between the scFv fragment of the first binding domain and the scFv fragment of the second binding domain is by any of the following means: (1) the VH of the first binding domain MUC1 The C-terminus of the second binding domain VH CD3 The N-terminus of the first binding domain is connected via the first linker sequence; (2) the VL MUC1 The C-terminus of the second binding domain VH CD3The N-terminus of the first binding domain is connected via the first linker sequence; (3) the VH MUC1 The C-terminus of the second binding domain VL CD3 The N-terminus of the first binding domain is connected via the first linker sequence; or (4) the VL MUC1 The C-terminus of the second binding domain VL CD3 The N-terminus of the peptide is connected via the first linker sequence.

[0016] In some embodiments, the connection between the scFv fragment of the second binding domain and the scFv fragment of the third binding domain is by any of the following means: (1) the VH of the second binding domain CD3 The C-terminus of the third binding domain VH CD28 The N-terminus of the second binding domain is connected via the second linker sequence; (2) the VL CD3 The C-terminus of the third binding domain VH CD28 The N-terminus of the second binding domain is connected via the second linker sequence; (3) the VH CD3 The C-terminus of the third binding domain VL CD28 The N-terminus of the second binding domain is connected via the second linker sequence; or (4) the VL CD3 The C-terminus of the third binding domain VL CD28 The N-terminus of the peptide is connected via the second linker sequence.

[0017] In some embodiments, the antigen binding protein comprises, in order from N-terminus to C-terminus: (1) VH of the first binding domain; MUC1 , a first linker sequence, a VL of the first binding domain MUC1 , a first linker sequence, a VH of the second binding domain CD3 , a first linker sequence, the second binding domain VL CD3 , a second linker sequence, a VH of the third binding domain CD28 , a first linker sequence, a VL of the third binding domain CD28 , or (2) the VL of the first binding domain MUC1 , a first linker sequence, a VH of the first binding domain MUC1 , a first linker sequence, a VH of the second binding domain CD3 , a first linker sequence, the second binding domain VL CD3 , a second linker sequence, a VH of the third binding domain CD28 , a first linker sequence, a VL of the third binding domain CD28 .

[0018] In some embodiments, the VH MUC1 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein the CDR-H1 sequence is shown in any one of SEQ ID NOs: 14, 20 and 26, the CDR-H2 sequence is shown in any one of SEQ ID NOs: 15, 21 and 27, and the CDR-H3 sequence is shown in any one of SEQ ID NOs: 16, 22 and 28.

[0019] In some embodiments, the VL MUC1 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein the CDR-L1 sequence is shown in any one of SEQ ID NOs: 17, 23 and 29, the CDR-L2 sequence is shown in any one of SEQ ID NOs: 18, 24 and 30, and the CDR-L3 sequence is shown in any one of SEQ ID NOs: 19, 25 and 31.

[0020] In some embodiments, the VH MUC1 The invention relates to a polynucleotide comprising the amino acid sequence shown in any one of SEQ ID NOs: 8, 10 and 12, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 8, 10 and 12.

[0021] In some embodiments, the VL MUC1 The invention relates to a polynucleotide comprising the amino acid sequence shown in any one of SEQ ID NOs: 9, 11 and 13, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 9, 11 and 13.

[0022] In some embodiments, the scFv fragment of the first binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-37, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 32-37.

[0023] In some embodiments, the VH CD3 It comprises a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein the CDR-H1 sequence is shown as SEQ ID NO: 42 or 48, the CDR-H2 sequence is shown as SEQ ID NO: 43 or 49, and the CDR-H3 sequence is shown as SEQ ID NO: 44 or 50.

[0024] In some embodiments, the VLCD3 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein the CDR-L1 sequence is shown as SEQ ID NO: 45 or 51, the CDR-L2 sequence is shown as SEQ ID NO: 46 or 52, and the CDR-L3 sequence is shown as SEQ ID NO: 47 or 53.

[0025] In some embodiments, the VH CD3 The amino acid sequence of SEQ ID NO: 38 or 40 is included, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 38 or 40.

[0026] In some embodiments, the VL CD3 The amino acid sequence of SEQ ID NO: 39 or 41 is included, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 39 or 41.

[0027] In some embodiments, the scFv fragment of the second binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 54-59, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 54-59.

[0028] In some embodiments, the VH CD28 It comprises a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein the CDR-H1 sequence is shown as SEQ ID NO: 62, the CDR-H2 sequence is shown as SEQ ID NO: 63, and the CDR-H3 sequence is shown as SEQ ID NO: 64.

[0029] In some embodiments, the VL CD28 It comprises a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein the CDR-L1 sequence is shown as SEQ ID NO: 65, the CDR-L2 sequence is shown as SEQ ID NO: 66, and the CDR-L3 sequence is shown as SEQ ID NO: 67.

[0030] In some embodiments, the VH CD28 The amino acid sequence of SEQ ID NO: 60 is included, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 60.

[0031] In some embodiments, the VLCD28 The amino acid sequence of SEQ ID NO: 61 is included, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 61.

[0032] In some embodiments, the scFv fragment of the third binding domain comprises the amino acid sequence shown in SEQ ID NO: 68 or 69, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in SEQ ID NO: 68 or 69.

[0033] In some embodiments, the first linker sequence comprises (G4S) n 、(G2S) n or (G2S) n GG, wherein n is an integer selected from 1 to 4.

[0034] In some embodiments, the first linker sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 1-4.

[0035] In some embodiments, the second linker sequence comprises the amino acid sequence of an IgD hinge region or a portion of an IgD hinge region.

[0036] In some embodiments, the second linker sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 5-7.

[0037] In some embodiments, the antigen binding protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 70-85.

[0038] In some embodiments, the antigen binding protein comprises four polypeptide chains I-IV, wherein the polypeptide chains I-IV constitute an IgG-like antibody structure, and the polypeptide chains I-IV respectively comprise the structures represented by the following formulas (I) to (IV): (I) and (II) VL MUC1 -CL MUC1 ; (III) VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD3 ; and (IV) VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD28 , wherein the VL MUC1 and CL MUC1 They are the immunoglobulin light chain variable region and light chain constant region that specifically bind to MUC1, respectively.

[0039] In some embodiments, the VL MUC1Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein the CDR-L1 sequence is shown in any one of SEQ ID NOs: 17, 23 and 29, the CDR-L2 sequence is shown in any one of SEQ ID NOs: 18, 24 and 30, and the CDR-L3 sequence is shown in any one of SEQ ID NOs: 19, 25 and 31.

[0040] In some embodiments, the VL MUC1 The invention relates to a polynucleotide comprising the amino acid sequence shown in any one of SEQ ID NOs: 9, 11 and 13, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 9, 11 and 13.

[0041] In some embodiments, the CL of polypeptide chain 1 MUC1 The domain comprises the amino acid sequence shown in SEQ ID NO:87.

[0042] In some embodiments, the polypeptide chain I and the polypeptide chain II each comprise an amino acid sequence selected from SEQ ID NO:88.

[0043] In some embodiments, the VH MUC1 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein the CDR-H1 sequence is shown in any one of SEQ ID NOs: 14, 20 and 26, the CDR-H2 sequence is shown in any one of SEQ ID NOs: 15, 21 and 27, and the CDR-H3 sequence is shown in any one of SEQ ID NOs: 16, 22 and 28.

[0044] In some embodiments, the VH MUC1 The invention relates to a polynucleotide comprising the amino acid sequence shown in any one of SEQ ID NOs: 8, 10 and 12, or an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 8, 10 and 12.

[0045] In some embodiments, the (scFv) CD3 The fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 54-59, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 54-59.

[0046] In some embodiments, the (scFv) CD28The fragment comprises the amino acid sequence shown in SEQ ID NO: 68 or 69, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in SEQ ID NO: 68 or 69.

[0047] In some embodiments, the polypeptide chain III comprises an amino acid sequence selected from SEQ ID NO: 89 or 90.

[0048] In some embodiments, the polypeptide chain IV comprises an amino acid sequence selected from SEQ ID NO:91.

[0049] In some embodiments, the CH3 domain portions of polypeptide chain III and polypeptide chain IV each comprise amino acid substitutions, and the amino acid substitutions in the CH3 domains of polypeptide chain III and polypeptide chain IV enable the two CH3 domains to form heterodimerization.

[0050] In some embodiments, wherein: (1) the CH3 domain of one of the polypeptide chain III and the polypeptide chain IV comprises amino acid substitutions at positions 297, 349, 356, 357, 366, 368, and 407 corresponding to human IgG1 according to the EU Index, the amino acid substitution at position 297 is N297A, the amino acid substitution at position 349 is Y349C, the amino acid substitution at position 356 is D356K, the amino acid substitution at position 357 is E357K, the amino acid substitution at position 366 is T366S, the amino acid substitution at position 368 is L368A, and the amino acid substitution at position 407 is Y407V; and (2) the CH3 domain of the other of the polypeptide chain III and the polypeptide chain IV comprises amino acid substitutions at positions 297, 349, 356, 357, 366, 368, and 407 corresponding to human IgG1 according to the EU Index. Index, comprising amino acid substitutions at positions 297, 354, 366, 370 and 439 corresponding to human IgG1, the amino acid substitution at position 297 is N297A, the amino acid substitution at position 354 is S354C, the amino acid substitution at position 366 is T366W, the amino acid substitution at position 370 is K370E, and the amino acid substitution at position 439 is K439E.

[0051] In some embodiments, the antigen binding protein comprises one of the following combinations of amino acid sequences: (1) polypeptide chains I and II respectively comprising the amino acid sequence of SEQ ID NO:88, polypeptide chain III comprising the amino acid sequence of SEQ ID NO:89, and polypeptide chain IV comprising the amino acid sequence of SEQ ID NO:91; or (2) polypeptide chains I and II respectively comprising the amino acid sequence of SEQ ID NO:88, polypeptide chain III comprising the amino acid sequence of SEQ ID NO:90, and polypeptide chain IV comprising the amino acid sequence of SEQ ID NO:91.

[0052] In another aspect, the present application provides a nucleic acid molecule encoding the antigen-binding protein described in the present application.

[0053] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the antigen binding protein described herein, which comprises a nucleotide sequence selected from any one of SEQ ID NOs: 92-107.

[0054] In some embodiments, the nucleic acid molecule comprises: a nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II described in the present application, and a nucleotide sequence encoding polypeptide chain III and polypeptide chain IV described in the present application; wherein: (1) the nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is as shown in SEQ ID NO: 109, the nucleotide sequence encoding polypeptide chain III is as shown in SEQ ID NO: 110, and the nucleotide sequence encoding polypeptide chain IV is as shown in SEQ ID NO: 112; or (2) the nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is as shown in SEQ ID NO: 109, the nucleotide sequence encoding polypeptide chain III is as shown in SEQ ID NO: 111, and the nucleotide sequence encoding polypeptide chain IV is as shown in SEQ ID NO: 112.

[0055] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0056] In some embodiments, the nucleic acid molecule is a DNA molecule and / or an RNA molecule.

[0057] In some embodiments, the RNA molecule is mRNA.

[0058] On the other hand, the present application provides a cell comprising the antigen binding protein described herein, the nucleic acid molecule described herein, and / or the vector described herein.

[0059] On the other hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein described herein and a pharmaceutically acceptable carrier.

[0060] In another aspect, the present application provides use of the antigen binding protein described herein in the preparation of a medicament for treating MUC1-positive tumors or cancers.

[0061] In another aspect, the present application provides a method for treating a MUC1-positive tumor or cancer, comprising administering to a patient in need thereof an effective amount of the antigen-binding protein, nucleic acid molecule, vector, cell, and / or pharmaceutical composition described herein.

[0062] In another aspect, the present application provides the antigen binding protein, nucleic acid molecule, vector, cell, and / or pharmaceutical composition described herein, for use in treating MUC1-positive tumors or cancers.

[0063] In some embodiments, the MUCl expression-positive tumor or cancer is selected from pancreatic cancer, ovarian cancer, breast cancer, lung adenocarcinoma, fallopian tube cancer, and colorectal cancer.

[0064] The multispecific antigen binding protein provided by the present application has at least the following advantages: (1) the antigen binding protein is a structurally complex co-activating CD3 + T-cell engagers (TCEs) have been shown to have ideal target-specific binding ability, T cell activation, T cell proliferation promotion, target cell killing activity and other positive effects on a variety of solid tumor cells, especially enhanced tumor infiltration; (2) The antigen binding protein has excellent effectiveness and successfully solved the difficult problem of T cell death after single signal activation in the field of solid tumor treatment, and can provide more lasting therapeutic effects, which is of significant significance for clinical application.

[0065] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0067] Figures 1A-1B Shown is a schematic diagram of the configuration of the trispecific antigen-binding protein of the present application.

[0068] Figures 2A-2E Shown is the binding activity of the trispecific antigen-binding protein of the present application to membrane-bound antigens.

[0069] Figures 3A-3F Shown is the effect of effector cell activation mediated by the trispecific antigen binding protein of the present application (PBMC system).

[0070] Figures 4A-4C Shown is a comparison of the T cell activation effects mediated by the trispecific antigen binding protein of the present application and other control antigen binding proteins (Jurkat-NFAT system); Figure 4D Shown is a schematic diagram of the configuration of a control trispecific antigen binding protein.

[0071] Figures 5A-5D Shown is a comparison of the killing activities of human PBMC against target cells mediated by the trispecific antigen-binding protein of the present application and other control antigen-binding proteins.

[0072] Figure 6 Shown is a comparison of the effector cell proliferation effects mediated by the trispecific antigen-binding protein of the present application and other control antigen-binding proteins.

[0073] Figures 7A-7F Shown are photos taken after the trispecific antigen-binding protein of the present application was co-incubated with T cells in a lung cancer organoid model.

[0074] Figure 8 Shown are the supernatant LDH data after the trispecific antigen binding protein of the present application was co-incubated with T cells in a lung cancer organoid model.

[0075] Figures 9A-9C Shown is the cytokine release of the trispecific antigen binding protein of the present application after co-incubation with T cells in a lung cancer organoid model.

[0076] Figures 10A-10D Shown are photos taken after the trispecific antigen-binding protein of the present application was co-incubated with T cells in an ovarian cancer organoid model.

[0077] Figure 11Shown are the supernatant LDH data after the trispecific antigen-binding protein of the present application was co-incubated with T cells in an ovarian cancer organoid model.

[0078] Figures 12A-12B Shown is the cytokine release of the trispecific antigen binding protein of the present application after co-incubation with T cells in an ovarian cancer organoid model.

[0079] Figures 13A-13C The results show the tumor-suppressing effect of the trispecific antigen-binding protein of the present application in a subcutaneously transplanted human (BXPC-3) pancreatic cancer-bearing model in mice.

[0080] Figures 14A-14C The results show the tumor-suppressing effect of the trispecific antigen-binding protein of the present application in a subcutaneously transplanted human (HCC1954) breast cancer tumor-bearing model in mice.

[0081] Figures 15A-15D The results show the anti-tumor effect of the trispecific antigen binding protein of the present invention in a mouse subcutaneously transplanted human (HPAC) pancreatic cancer tumor model. DETAILED DESCRIPTION

[0082] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0083] Definition of terms

[0084] In this application, the term "T cell engager (TCE)" is generally a class of artificial bispecific or multispecific antibodies based on antibodies or antibody fragments, which can connect T cells in the body to cells expressing specific tumor-targeting related proteins. In some embodiments, TCE is a bispecific antibody, also known as a "bispecific T cell engager" or BiTE (Bispecific T-cell Engager), which has two different target antigen binding domains. One side of the binding domain can recognize tumor-associated antigens (such as CD19, CD33, BCMA, etc.), and the other side usually specifically recognizes the CD3 / T cell receptor (TCR) complex, thereby connecting T cells and tumor target cells, causing T cell activation and killing tumor target cells. The term "CD3+TCE" generally refers to antibodies that connect CD3 on the surface of T cells and specific antigens on the surface of tumor cells. It can recruit and activate polyclonal T cell populations near the tumor to kill and lyse tumor cells. But in some cases, the initial T lymphocytes need the synergistic effect of two activation signals from rest to full activation. This is because after the antigen / MHC complex is recognized by TCR, the signal conducted by the CD3-TCR complex is not enough to fully activate the T cell, and the signal generated by the costimulatory molecule is needed as a supplement. As used in this application, "co-activation CD3+TCE" refers to the multispecific antibodies that simultaneously achieve the targeting of tumor cells and the "double signal" (the first signal comes from CD3 / TCR recognition MHC / antigen peptide complex; the second signal is provided by the costimulatory molecule, which is a co-stimulatory signal) activation of T cells. For example, the B7-CD28 complex formed by the non-covalent binding of CD28 molecules and the ligand (B7) on antigen presenting cells (APC) can work with CD3 antibodies to exert costimulatory effects, stimulate T cell differentiation, proliferation and the secretion of cytokines such as IL-2, and play an important role in tumor immunity, autoimmune disease treatment and immune intervention against transplant rejection.

[0085] In this application, the term "antigen binding protein" generally refers to a polypeptide construct derived from an antibody that is capable of specifically binding to an antigen but does not contain all the elements of a natural antibody. In particular, an antigen binding fragment does not contain some or all of the constant domains of an antibody and may contain only one, rather than two, antigen binding sites. In certain embodiments of the present application, the term "multispecific antigen binding protein" may be used interchangeably with "multispecific antibody," which generally refers to an antibody or antibody analog that is capable of simultaneously and specifically binding to multiple targets, i.e., an antibody construct having one or more binding sites that bind to a first epitope and one or more binding sites that bind to a second epitope, and optionally even other binding sites that bind to other epitopes. For example, a "trispecific antibody" refers to an antibody or antibody analog that is capable of simultaneously and specifically binding to three targets.

[0086] In this application, the term "antibody" refers in particular to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain constant region comprises three or (in the case of IgM- or IgE-type antibodies) four heavy chain constant domains (CH1, CH2, CH3 and CH4), wherein the first constant domain CH1 is adjacent to the variable region and can be connected to the second constant domain CH2 by a hinge region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)), wherein each variable region comprises three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region can be of any type, such as a γ-, δ-, α-, μ- or ε-type heavy chain. Preferably, the heavy chain of the antibody is a γ chain. In addition, the light chain constant region can also be of any type, such as a κ- or λ-type light chain. Preferably, the light chain of the antibody is a κ chain. The terms "γ- (δ-, α-, μ- or ε-) type heavy chain" and "κ- (λ-) type light chain" refer to an antibody heavy chain or antibody light chain having an amino acid sequence of a heavy chain or light chain constant region derived from a naturally occurring, especially a human heavy chain or light chain constant region amino acid sequence. In particular, the amino acid sequence of the constant domain of a γ-type (especially γ1-type) heavy chain is at least 95%, in particular at least 98%, identical to the amino acid sequence of the constant domain of a human γ (especially one of the allotypes of human γ1) antibody heavy chain. In addition, the amino acid sequence of the κ-type light chain constant domain is in particular at least 95%, in particular at least 98% identical to the amino acid sequence of the constant domain of one of the allotypes of a human κ antibody light chain. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system. The antibodies may be, for example, humanized, human, or chimeric antibodies.

[0087] The antigen-binding portion of an antibody generally refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments of an antibody include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region, each Fab fragment binding to the same antigen; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a dAb fragment consisting of the VH domain.

[0088] In this application, the term "specific binding" generally refers to the binding of an agent, such as an antibody, to a target, such as an epitope, for which it is specific, more strongly than to another target. d ) is lower than the dissociation constant of the second target, then its binding to the first target is stronger than that to the second target. d ) is generally used to reflect the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody or chimeric antibody immune effector cell adaptor) and its binding partner (e.g., an antigen), i.e., "binding affinity". Affinity can be measured by common methods known in the art (including those described in the examples of this application). Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens faster and tend to remain bound for a longer time. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this application. Preferably, the dissociation constant of the target to which the agent specifically binds is more than 100 times, 200 times, 500 times, or more than 1000 times lower than the dissociation constant of the target to which the agent does not specifically bind; alternatively, in certain embodiments, the antigen-binding protein that specifically binds to the target has the following dissociation constant (K d ): ≦1μM, ≦100nM, ≦10nM, ≦1nM or ≦0.1nM. In addition, the dissociation constant K d The reciprocal affinity constant K a It can also reflect binding affinity, which specifically refers to the strength of the binding between the antibody and the antigen. Thus, the term "specific binding" can also refer to a binding affinity between binding partners of at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 The affinity constant K a Antibodies that are specific for an antigen are specifically antibodies that can 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 K a For example, in certain embodiments of the present application, an antibody that specifically binds to MUC1 is preferably able to bind to the antigen with an affinity of at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 Ka The affinity of the protein binds to MUC1.

[0089] In this application, the term "MUC1" generally refers to the protein MUC1, also known as mucin-1, polymorphic epithelial mucin (PEM), or cancer antigen 15-3, particularly human MUC1. MUC1 is a member of the mucin family and encodes a membrane-bound, glycosylated mucin. MUC1 has a core protein mass of 120-225 kDa, which is increased to 250-500 kDa by glycosylation. It extends 200-500 nanometers beyond the cell surface. The protein is anchored to the apical surface of many epithelial cells via a transmembrane domain. The extracellular domain includes a variable number of tandem repeats (VNTR) domain of 20 amino acids, where the number of repeats varies from 20 to 120 in different individuals. These repeats are rich in serine, threonine, and proline residues, which allow for heavy O-glycosylation. In certain embodiments of the application, the term "MUC1" may refer to tumor-associated MUC1 ("TA-MUC1"). TA-MUC1 is MUC1 present on cancer cells. This MUC1 differs from the MUC1 present on non-cancerous cells by its much higher expression level, its localization, and its glycosylation. Specifically, TA-MUC1 is nonpolarized and present throughout the cell surface of cancer cells, whereas in non-cancerous cells, MUC1 has strictly apical expression, which often serves as one of the criteria for distinguishing tumor-associated MUC1 from normal MUC1. Furthermore, TA-MUC1 has aberrant O-glycosylation that exposes novel peptide epitopes on the MUC1 protein backbone and novel carbohydrate tumor antigens, such as the Thomsen-Friedenreich antigen α (TFα), also known as Core-1, which refers to the disaccharide Gal-β1,3-GalNAc, which in cancer cells is O-glycosidically linked to the hydroxyamino acids serine or threonine of proteins in the α-isomer configuration.

[0090] In this application, the term "CD3" generally refers to the T cell co-receptor CD3 (cluster of differentiation 3), which is a transmembrane protein whose transmembrane region is connected to the transmembrane region of the two peptide chains of TCR through a salt bridge to form a TCR-CD3 complex, which jointly participates in the recognition of antigens by T cells and transduces the generated activation signals into T cells. Unless otherwise specified, the CD3 specifically bound by the second binding domain in the context of this application is human CD3. 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.

[0091] In this application, the term "CD28" generally refers to a dimeric transmembrane glycoprotein that is present in approximately 80% of human CD4 +T cells and 50% of CD8 + CD28 is expressed on the surface of T cells. It is a T cell co-stimulatory molecule that binds to its ligands CD80 (B7-1) or CD86 (B7-2) and amplifies the primary signal transmitted by TCR / CD3, maintaining T cell survival and promoting cytokine-induced T cell proliferation and differentiation. Unless otherwise specified, the CD28 specifically bound by the third binding domain in the context of this application is human CD28.

[0092] In this application, the term "epitope" generally refers to any polypeptide determinant that is capable of specifically binding to a target antigen. In certain embodiments, epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope can be an antigen or a region of an antigen to which a binding protein binds. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, when the equilibrium dissociation constant is ≤10 -8 M, more preferably when the equilibrium dissociation constant is ≤10 -9 M, and most preferably when the dissociation constant is ≤10 -10 When M, the binding protein is said to specifically bind to the antigen.

[0093] In this application, the term "binding domain" in the context of a multispecific antigen-binding protein refers to a polypeptide structure that can specifically recognize and bind to a target, and is sometimes referred to as a "domain".

[0094] In this application, the term "scFv" generally refers to a single chain variable region fragment (single chain fragment variable), sometimes also referred to as a single chain antibody, which is a fusion protein consisting of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody. In scFv, the connection order of VH and VL can be swapped, and VH can be placed at the N-terminus or VL can be placed at the N-terminus. VH and VL can be connected by a "linker" sequence. The single chain variable region fragment can also include a heavy chain variable region fragment (Single variable domain on a heavy chain (VHH)), or nanobody for short. Due to the lack of light chain, nanobody has only 3 antigen recognition regions (CDR regions) belonging to the heavy chain. The molecular weight is about one-tenth of that of a traditional antibody and about one-half of that of an antigen binding fragment (scFV, VH-VL).

[0095] In this application, a "linker sequence" generally refers to a peptide segment used to connect two binding domains, and also refers to a peptide segment used to connect VH and VL in a binding domain. A "first linker sequence" specifically refers to a linker used to connect the first binding domain and the second binding domain, and a "second linker sequence" specifically refers to a linker used to connect the second binding domain and the third binding domain. The first linker sequence and the second linker sequence are both linkers used to connect different binding domains, and are therefore collectively referred to as "inter-domain linkers." If a linker also exists between the VH and VL contained in a domain, it is referred to as an "intra-domain linker."

[0096] In this application, the term "G4S" refers to Gly-Gly-Gly-Gly-Ser, a pentapeptide unit consisting of four glycine amino acids and one serine amino acid, one or more of which are represented in this application as (G4S) n ) is a commonly used linker sequence in recombinant fusion proteins. Similarly, "G2S" refers to Gly-Gly-Ser.

[0097] In this application, the term "homology" or "identity" is generally used to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percent identity". Preferably, it is determined according to the present invention over the entire length of a reference sequence or over the entire length of the corresponding portion of the reference sequence (which corresponds to the sequence defining homology or identity). Antibodies derived from parent antibodies (which are defined by one or more amino acid sequences (e.g., specific CDR sequences or specific variable region sequences)) are particularly antibodies having amino acid sequences, such as CDR sequences or variable region sequences, that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous or identical, especially identical, to each amino acid sequence of the parent antibody. In certain embodiments, the antibody derived from the parent antibody (i.e., its derivative) comprises the same CDR sequences as the parent antibody, but differs in the remaining sequences of the variable region. The percent homology of two sequences can be calculated as follows: after aligning the two sequences, the number of positions where the residues are identical is divided by the total length of the aligned sequences and multiplied by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, for example, the BLAST suite available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410).

[0098] In the present application, the term "IgD" refers to one of the isotypes of immunoglobulins, which has a Y-shaped structure like IgG. In certain embodiments of the present application, a fragment of the hinge region of IgD is used as a linker sequence.

[0099] As used herein, the term "heterodimerization" generally refers to the formation of a dimeric complex by binding two polypeptides via a specific domain, wherein the two polypeptides have amino acid sequences that differ by at least one amino acid residue at corresponding positions, where the corresponding positions are determined according to the Kabat EU index numbering system. The complex may further comprise other polypeptides, which are also covalently or non-covalently bound to the remaining polypeptides. For example, in certain embodiments of the present application, the dimeric complex comprises two or four polypeptides.

[0100] In this application, the term "nucleic acid" may include single-stranded and double-stranded nucleic acids and ribonucleic acids and deoxyribonucleic acids. It may include naturally occurring and synthetic nucleotides, and may be naturally or synthetically modified, for example, by methylation, 5'- and / or 3'-capping. In this application, the term "nucleic acid encoding ..." or "(nucleotide) sequence encoding ..." generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0101] In the present application, the term "vector" generally refers to any molecule (for example, nucleic acid, plasmid or virus, virus-like particle, polycation, peptide vector, liposome and / or hybrid vector) for transferring coding information to a host cell. The term "vector" includes a nucleic acid molecule that can transship another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule that can insert an extra DNA segment. Another type of vector is a viral vector, in which an extra DNA segment can be inserted into the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with a bacterial origin of replication) in the host cell introducing them. In addition, in some instances, "liposomes" or "lipid nanoparticles (LNP)" are also commonly used in delivering DNA molecules and / or RNA molecules (for example, DNA or mRNA encoding the antigen-binding proteins of the present application). Among them, liposomes generally refer to vesicles with an internal space that are isolated from the external medium by one or more bilayer membranes. They are biocompatible and non-toxic, and can therefore deliver hydrophilic and lipophilic drug molecules, protect their cargo from being degraded by plasma enzymes, and transport their loads across biological membranes and the blood-brain barrier (BBB). In some cases, LNPs can also be used to deliver RNP complexes. LNPs generally refer to particles containing multiple (i.e., more than one) lipid molecules that are physically bound to each other by intermolecular forces (e.g., covalently or non-covalently); for example, when LNPs are used to deliver the nucleic acid molecules of the present application, they can encapsulate the nucleic acid in cationic lipid particles (e.g., liposomes) and can relatively easily deliver the nucleic acid molecules to cells. Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell and thus replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are generally referred to as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used form of vector. However, this disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0102] In the present application, the terms "cell" and "host cell" are used interchangeably, and generally refer to cells containing exogenous recombinant DNA such as expression vectors, and also refer to cells containing polypeptides or proteins obtained by recombinant DNA expression. Such host cells can be used for the production of the trispecific antibodies of the present application. In certain embodiments, as a host cell, there are no particular restrictions as long as it is a host cell that can be used for the expression of polypeptide sequences such as the trispecific antibodies of the present application. Recombinant host cells or host cells refer not only to specific subject cells, but also to the offspring of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from the parental cells, but such cells are still included in the scope of the terms "cell" or "host cell" used in the present application.

[0103] In this application, the term "pharmaceutical composition" generally refers to a compound or composition that can induce a desired therapeutic effect when properly administered to a patient. Preferably, the pharmaceutical composition comprises the drug (e.g., antigen binding protein or nucleic acid molecule) of the present application or its variant, prodrug or other biologically active form, and a carrier, diluent or pharmaceutical excipient, such as a buffer, preservative and tonicity regulator.

[0104] As used herein, the term "pharmaceutically acceptable carrier" generally refers to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

[0105] In this application, the terms "effective amount" and "therapeutically effective amount" are used interchangeably, and generally refer to an amount or dosage sufficient to produce a desired therapeutic outcome when a pharmaceutical composition comprising one or more binding proteins is administered. More specifically, a therapeutically effective amount is sufficient to treat a specified condition, illness or disease over a period of time, and specifically may refer to the amount of a drug (e.g., antigen-binding proteins provided herein) or a pharmaceutical composition thereof that improves, alleviates, mitigates and / or delays one or more of its symptoms. In some examples for cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other cells that do not wish to proliferate. 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 may be administered in one or more administrations. In some embodiments, an effective amount of a drug or pharmaceutical composition thereof can: (1) reduce the number of cancer cells; (2) reduce the size of a tumor; (3) inhibit, delay, slow down, and preferably prevent to some extent the infiltration of cancer cells into surrounding organs; (4) inhibit (i.e., slow down or stop to some extent) tumor metastasis; (5) inhibit tumor growth; (6) prevent or delay the occurrence and / or recurrence of a tumor; (7) alleviate to some extent one or more symptoms associated with cancer. The effective amount can vary depending on the specific antigen binding protein used and also depends on a variety of factors related to the patient being treated and the severity of the condition. For example, if the antigen binding protein is to be administered in vivo, factors such as the patient's age, weight, and health, as well as dose-response curves and toxicity data obtained in preclinical animal work, would be among the factors considered. Determining an effective amount or therapeutically effective amount for a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0106] In this application, the term "treatment" generally refers to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include those with a condition as well as those susceptible to a condition or those in whom a condition is to be prevented. In specific embodiments, the binding proteins can be used to treat a human with cancer, or a human susceptible to cancer, or to ameliorate cancer in a human subject. The binding proteins can also be used to prevent cancer in a human patient.

[0107] In this application, the term "tumor" generally refers to a group of cells or tissues formed by misregulated cell proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with normal tissues and usually form a distinct tissue mass, which can be benign or malignant.

[0108] In the present application, the term "cancer" particularly includes leukemia, seminoma, melanoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer and lung cancer and metastases thereof. The term cancer according to the present application also includes cancer metastases.

[0109] In this application, the term "patient" generally can include humans, non-human primates (e.g., monkeys) or other animals, particularly mammals, such as cows, horses, pigs, sheep, goats, dogs, cats or rodents, such as mice and rats. In particularly preferred embodiments, the patient is a human.

[0110] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0111] In this application, the term "selected from" generally refers to the selected objects and all combinations thereof. For example, "selected from A, B and C" means all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C, or A+B+C.

[0112] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation 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 specified value. Detailed Description of the Invention

[0114] In one aspect, the present application provides a multispecific antigen-binding protein comprising (1) a first binding domain capable of specifically binding to human tumor-associated mucin-1 (MUC1), wherein the first binding domain comprises a heavy chain variable region (VH MUC1 ) and light chain variable region (VL MUC1 ); (2) a second binding domain capable of specifically binding to a human CD3 polypeptide, wherein the second binding domain comprises a heavy chain variable region (VH CD3 ) and light chain variable region (VL CD3 ); and (3) a third binding domain capable of specifically binding to a human CD28 polypeptide, wherein the third binding domain comprises a heavy chain variable region (VH CD28 ) and light chain variable region (VL CD28 ).

[0115] Linking of the first, second and third binding domains :

[0116] In some embodiments, the first binding domain, the second binding domain, and the third binding domain of the multispecific antigen-binding protein described herein are all single-chain antibody (scFv) fragments.

[0117] Specifically, the scFv of the first binding domain may comprise VH MUC1 , the first linker sequence and VL MUC1 , or VL can be included from N-terminus to C-terminus MUC1 , the first linker sequence and VH MUC1 The scFv of the second binding domain may comprise VH CD3 , the first linker sequence and VL CD3 , or VL can be included from N-terminus to C-terminus CD3 , the first linker sequence and VH CD3 The scFv of the third binding domain may comprise VH from N-terminus to C-terminus CD28 , the first linker sequence and VL CD28 , or VL can be included from N-terminus to C-terminus CD28 , the first linker sequence and VH CD28 .

[0118] In the above situation, the scFv of the first binding domain and the scFv of the second binding domain are connected via a first linker sequence, which can specifically produce the following domain connection combinations: in the order of connection from N-terminus to C-terminus, VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VH CD3 -First linker sequence-VL CD3 、VL MUC1 -First linker sequence-VH MUC1 -First linker sequence-VH CD3 -First linker sequence-VL CD3 、VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3 , or VL MUC1 -First linker sequence-VH MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3In the same situation as above, the scFv of the second binding domain is connected to the scFv of the third binding domain via a second linker sequence, which can specifically produce the following domain connection combinations: in the order of connection from N-terminus to C-terminus, VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 、VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 、VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 , or VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 .

[0119] Furthermore, the antigen binding protein comprises, in order from N-terminus to C-terminus: a scFv of a first binding domain, a first linker sequence, a scFv of a second binding domain, a second linker sequence, and a scFv of a third binding domain; specifically, the following domains can be connected to form a combination: (1) VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 ; (2) VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 ; (3) VL MUC1 -First linker sequence-VH MUC1 -First linker sequence-VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 ; (4) VL MUC1 -First linker sequence-VH MUC1-First linker sequence-VH CD3 -First linker sequence-VL CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 ; (5) VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 ; (6) VH MUC1 -First linker sequence-VL MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 ; (7) VL MUC1 -First linker sequence-VH MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VH CD28 -First linker sequence-VL CD28 ; or (8) VL MUC1 -First linker sequence-VH MUC1 -First linker sequence-VL CD3 -First linker sequence-VH CD3 -Second linker sequence-VL CD28 -First linker sequence-VH CD28 .

[0120] In the above embodiment, the VH MUC1Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence. The CDR-H1 sequence may be any one of NYWMH (SEQ ID NO: 14), DHAIH (SEQ ID NO: 20) and NYWMN (SEQ ID NO: 26); the CDR-H2 sequence may be any one of YINPSSGYTQYNQKFKD (SEQ ID NO: 15), HFSPGNTDIKYNDKFKG (SEQ ID NO: 21) and EIRLKSNNYTTHYAESVKG (SEQ ID NO: 27); and the CDR-H3 sequence may be any one of YYGDYLFPY (SEQ ID NO: 16), STFFFDY (SEQ ID NO: 22) and HYYFDY (SEQ ID NO: 28). Specifically, the VH MUC1 It may comprise: (1) CDR-H1 sequence NYWMH (SEQ ID NO: 14), CDR-H2 sequence YINPSSGYTQYNQKFKD (SEQ ID NO: 15), and CDR-H3 sequence YYGDYLFPY (SEQ ID NO: 16); or (2) CDR-H1 sequence DHAIH (SEQ ID NO: 20), CDR-H2 sequence HFSPGNTDIKYNDKFKG (SEQ ID NO: 21), and CDR-H3 sequence STFFFDY (SEQ ID NO: 22); or (3) CDR-H1 sequence NYWMN (SEQ ID NO: 26), CDR-H2 sequence EIRLKSNNYTTHYAESVKG (SEQ ID NO: 27), and CDR-H3 sequence HYYFDY (SEQ ID NO: 28). For example, the VH MUC1 It may comprise any of the following amino acid sequences:

[0121] (a) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWMHWVRQAPGQGLEWMGYINP SSGYTQYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCSTYYGDYLFPYWGQG TTVTVSS (SEQ ID NO: 8);

[0122] (b) EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEVVMGHFSPG NTDIKYNDKFKGQVTLSVDRSINTAYLQWSSLKASDTAIYFCKTSTFFFDYWGQGTRVTV SS (SEQ ID NO: 10); and

[0123] (c) EVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMNWVRQAPGKGLEWVGEIRL KSNNYTTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFDYWGQGTL VTVSS (SEQ ID NO: 12).

[0124] Alternatively, the VH MUC1 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 8, 10, and 12, and the differences in the amino acid sequences are all in the non-CDR regions.

[0125] In the above embodiment, the VL MUC1 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Wherein, the CDR-L1 sequence may be any one of RSSQDIVYGNGNTYLE (SEQ ID NO: 17), KSSQSLLNSGDQKNYLT (SEQ ID NO: 23) and RSSKSLLHSNGITYF (SEQ ID NO: 29); the CDR-L2 sequence may be any one of KVSNRFS (SEQ ID NO: 18), WASTRES (SEQ ID NO: 24) and QMSNLAS (SEQ ID NO: 30); and the CDR-L3 sequence may be any one of FQGSHVPYT (SEQ ID NO: 19), QNDYSYPLT (SEQ ID NO: 25) and AQNLELPPT (SEQ ID NO: 31). Specifically, the VL MUC1It may comprise: (1) CDR-L1 sequence RSSQDIVYGNGNTYLE (SEQ ID NO: 17), CDR-L2 sequence KVSNRFS (SEQ ID NO: 18), and CDR-L3 sequence FQGSHVPYT (SEQ ID NO: 19); or (2) CDR-L1 sequence KSSQSLLNSGDQKNYLT (SEQ ID NO: 23), CDR-L2 sequence WASTRES (SEQ ID NO: 24), and CDR-L3 sequence QNDYSYPLT (SEQ ID NO: 25); or (3) CDR-L1 sequence RSSKSLLHSNGITYF (SEQ ID NO: 29), CDR-L2 sequence QMSNLAS (SEQ ID NO: 30), and CDR-L3 sequence AQNLELPPT (SEQ ID NO: 31). For example, the VL MUC1 It may comprise any of the following amino acid sequences:

[0126] (a) DVVMTQSPLSLPVTLGQPASISCRSSQDIVYGNGNTYLEWYLQRPGQSPRLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIK (SEQ ID NO: 9);

[0127] and

[0128] (c) DIVMTQSPLSNPVTPGEPASISCRSSKSLLHSNGITYFFWYLQKPGQSPQLLIYQMS NLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGQGTKVEIKR (SEQ ID NO: 13).

[0129] Alternatively, the VL MUC1It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 9, 11, and 13, and the differences in the amino acid sequences are all in the non-CDR regions.

[0130] Therefore, according to the above situation, when the scFv of the first binding domain can comprise VH MUC1 , the first linker sequence and VL MUC1 , or VL can be included from N-terminus to C-terminus MUC1 , the first linker sequence and VH MUC1 The following table shows, in a non-limiting manner, exemplary amino acid sequences that the scFv of the first binding domain may comprise:

[0131]

[0132]

[0133] Alternatively, in certain embodiments, the scFv of the first binding domain may comprise VH MUC1 , the first linker sequence and VL MUC1 , or VL can be included from N-terminus to C-terminus MUC1 , the first linker sequence and VH MUC1 When the scFv of the first binding domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or homologous to any one of the amino acid sequences shown in the table above, and the differences in the amino acid sequences are all in the non-CDR regions.

[0134] In the above embodiment, the VH CD3Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence. Wherein, the CDR-H1 sequence may be RYTMH (SEQ ID NO: 42) or TYAMN (SEQ ID NO: 48); the CDR-H2 sequence may be YINPSRGYTNYNQKFKD (SEQ ID NO: 43) or RIRSKYNNYATYYAAPVKG (SEQ ID NO: 49); and the CDR-H3 sequence may be YYDDHYCLDY (SEQ ID NO: 44) or HGNFGNSYVSWFAY (SEQ ID NO: 50). Specifically, the VH CD3 It may comprise: (1) CDR-H1 sequence RYTMH (SEQ ID NO: 42), CDR-H2 sequence YINPSRGYTNYNQKFKD (SEQ ID NO: 43), and CDR-H3 sequence YYDDHYCLDY (SEQ ID NO: 44); or (2) CDR-H1 sequence TYAMN (SEQ ID NO: 48), CDR-H2 sequence RIRSKYNNYATYYAAPVKG (SEQ ID NO: 49), and CDR-H3 sequence HGNFGNSYVSWFAY (SEQ ID NO: 50). For example, the VH CD3 It may comprise any of the following amino acid sequences:

[0135] and

[0136] (b) EVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSK YNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWF AYWGQGTTVTVSS (SEQ ID NO: 40).

[0137] Alternatively, the VH CD3It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 38 or 40, and the differences in the amino acid sequences are all in the non-CDR regions.

[0138] In the above embodiment, the VL CD3 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Wherein, the CDR-L1 sequence may be RASSSVSYMN (SEQ ID NO: 45) or RSSTGAVTTSNYAN (SEQ ID NO: 51); the CDR-L2 sequence may be DTSKVAS (SEQ ID NO: 46) or GTNKKAS (SEQ ID NO: 52); and the CDR-L3 sequence may be QQWSSNPLT (SEQ ID NO: 47) or ALWYSNLWV (SEQ ID NO: 53). Specifically, the VL CD3 It may comprise: (1) CDR-L1 sequence RASSSVSYMN (SEQ ID NO: 45), CDR-L2 sequence DTSKVAS (SEQ ID NO: 46), and CDR-L3 sequence QQWSSNPLT (SEQ ID NO: 47); or (2) CDR-L1 sequence RSSTGAVTTSNYAN (SEQ ID NO: 51), CDR-L2 sequence GTNKKAS (SEQ ID NO: 52), and CDR-L3 sequence ALWYSNLWV (SEQ ID NO: 53). For example, the VL CD3 It may comprise any of the following amino acid sequences:

[0139] (a) DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 39); and

[0140] (b) QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAFRGLIGGTNK KASGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 41).

[0141] Alternatively, the VL CD3 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 39 or 41, and the differences in the amino acid sequences are all in the non-CDR regions.

[0142] Therefore, according to the above situation, when the scFv of the second binding domain can comprise VH CD3 , the first linker sequence and VL CD3 , or VL can be included from N-terminus to C-terminus CD3 , the first linker sequence and VH CD3 The following table shows, in a non-limiting manner, exemplary amino acid sequences that the scFv of the second binding domain may comprise, wherein the region shown in italics is an alternative first linker sequence, which may be selected from the first linker sequences provided herein:

[0143]

[0144]

[0145] Alternatively, in certain embodiments, the scFv of the second binding domain may comprise VH CD3 , the first linker sequence and VL CD3 , or VL can be included from N-terminus to C-terminus CD3 , the first linker sequence and VH CD3When the scFv of the second binding domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or homologous to any one of the amino acid sequences shown in the table above, and the differences in the amino acid sequences are all in the non-CDR regions.

[0146] In the above embodiment, the VH CD28 comprising a CDR-H1 sequence, a CDR-H2 sequence, and a CDR-H3 sequence. The CDR-H1 sequence may be SYYIH (SEQ ID NO: 62); the CDR-H2 sequence may be CIYPGNVNTNYNEKFKD (SEQ ID NO: 63); and the CDR-H3 sequence may be SHYGLDWNFDV (SEQ ID NO: 64). Specifically, the VH CD28 It may comprise: CDR-H1 sequence SYYIH (SEQ ID NO: 62), CDR-H2 sequence CIYPGNVNTNYNEKFKD (SEQ ID NO: 63), and CDR-H3 sequence SHYGLDWNFDV (SEQ ID NO: 64). For example, the VH CD28 It may contain the following amino acid sequence:

[0147] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNV NTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTT VTVSS (SEQ ID NO: 60).

[0148] Alternatively, the VH CD28 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 60, and the differences in the amino acid sequences are all in the non-CDR regions.

[0149] In the above embodiment, the VL CD28Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Wherein, the CDR-L1 sequence may be HASQNIYVWLN (SEQ ID NO: 65); the CDR-L2 sequence may be KASNLHT (SEQ ID NO: 66); and the CDR-L3 sequence may be QQGQTYPYT (SEQ ID NO: 67). Specifically, the VL CD28 It may comprise: CDR-L1 sequence HASQNIYVWLN (SEQ ID NO: 65), CDR-L2 sequence KASNLHT (SEQ ID NO: 66), and CDR-L3 sequence QQGQTYPYT (SEQ ID NO: 67). For example, the VL CD28 It may contain the following amino acid sequence:

[0150] DIQMTQSPSSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR (SEQ ID NO: 61).

[0151] Alternatively, the VL CD28 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 61, and the differences in the amino acid sequences are all in the non-CDR regions.

[0152] Therefore, according to the above situation, when the scFv of the third binding domain can comprise VH CD28 , the first linker sequence and VL CD28 , or VL can be included from N-terminus to C-terminus CD28 , the first linker sequence and VH CD28 When, the following table shows, in a non-limiting manner, exemplary amino acid sequences that the scFv of the third binding domain may comprise:

[0153]

[0154] Alternatively, in certain embodiments, the scFv of the third binding domain may comprise VH CD28 , the first linker sequence and VLCD28 , or VL can be included from N-terminus to C-terminus CD28 , the first linker sequence and VH CD28 When the scFv of the third binding domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or homologous to any one of the amino acid sequences shown in the table above, and the differences in the amino acid sequences are all in the non-CDR regions.

[0155] On the basis of determining each binding domain, the linker used to connect the various binding domains, as well as the linker used to connect the VH and VL in each binding domain, are also crucial for the structure and function of the trispecific antibody, especially the former. On the one hand, the linker sequence and the functional domain together constitute the entire trispecific antibody molecule, and the linker sequence will affect the structural stability of the entire trispecific antibody molecule. On the other hand, the length and flexibility of the linker sequence will affect the spatial position relationship between the various binding domains, and thus affect the binding of each domain to its target. In addition, whether certain amino acids in the linker sequence are prone to further modification, such as dimerization or glycosylation, will also affect the overall properties of the trispecific antibody molecule. The trispecific antibody provided by the present application has a first binding domain that targets tumor cells, while the second and third binding domains target immune cells. The adjacent first and second binding domains will inevitably bind to different cells. In this case, the first domain and the second domain need to have a certain ability to move relative to each other, and when binding to different cells, the position can be adjusted to make targeted binding easier. Thus, in all the above embodiments, the first linker sequence can be a flexible linker, specifically selected from a GS linker peptide, wherein G is glycine (Gly) and S is serine (Ser). More specifically, the first linker sequence can be represented as comprising (G4S) n 、(G2S) n or (G2S) nGG, wherein n is an integer selected from 1 to 4. For example, the first linker sequence can be: GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS (SEQ ID NO: 1), GGGGSGGGGSGGGGSGGGGS, GGS, GGSGGS, GGSGGSGGSGGS, GGSGGSGGSGGS (SEQ ID NO: 3), or GGSGGSGGSGGSGGSGG (SEQ ID NO: 2). Similarly, the linker for connecting VH and VL in each binding domain of the trispecific antibody provided by the present application can also be selected from the above-mentioned first linker sequence.

[0156] In the above-mentioned embodiment, the second linker sequence is used to connect the second and third binding domains, both of which bind to receptors on the surface of T cells. To prevent steric hindrance caused by binding of one domain from affecting the binding of the other domain to the same T cell, a slightly longer and more rigid second linker sequence is more advantageous for avoiding steric hindrance. For example, the second linker sequence can be selected from the amino acid sequence comprising an IgD hinge region, an amino acid sequence of a portion of an IgD hinge region, and variants thereof. Furthermore, the amino acid sequence of the portion of the IgD hinge region that can serve as the second linker sequence can be obtained by truncating the amino acid sequence of the IgD hinge region to a fragment containing at least one, at least two, at least three, at least four, or at least five potential glycosylation sites. In other embodiments, the amino acid sequence variant of the IgD hinge region or a partial IgD hinge region that can serve as the second linker sequence is obtained by removing or replacing cysteines that may contribute to dimer formation and / or shortening the overall length of the hinge region. For example, the second linker sequence can be RNTGRGGEEKKKEKEKEEQEERETKTPESPSHTQPLGV (SEQ ID NO: 5), RNTGRGGEEKKKEKEKEEQEEKETKTPESPSHTQPLGV (SEQ ID NO: 6), or RNTGKGGEEKKKEKEKEEQEERETKTPESPSHTQPLGV (SEQ ID NO: 7).

[0157] In summary of the above embodiments, the multispecific antigen-binding protein described in the present application may comprise an amino acid sequence as shown in any of the following:

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] In some embodiments, the multispecific antigen-binding protein described herein comprises four polypeptide chains I-IV, wherein the polypeptide chains I-IV constitute an IgG-like antibody structure and respectively comprise the structures represented by the following formulae (I) to (IV): (I) and (II) VL MUC1 -CL MUC1 ; (III) VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD3 ; and (IV) VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD28 , wherein the VL MUC1 and CL MUC1 In the present application, the IgG-like antibody structure specifically refers to the heterodimer formed by the four polypeptide chains I-IV, wherein the VL of polypeptide chain I MUC1 VH with polypeptide chain III MUC1 Forming the first binding domain capable of specifically binding to MUC1, the VL of the polypeptide chain II MUC1 With the polypeptide chain IV VH MUC1 Forming another first binding domain that can specifically bind to MUC1; targeting of CD3 and CD28 is achieved by connecting to the C-terminus of polypeptide chain III and polypeptide chain IV (immunoglobulin Fc region) (scFv) CD3 and (scFv) CD28 achieved.

[0167] In the above embodiment, the VL MUC1Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Wherein, the CDR-L1 sequence may be any one of RSSQDIVYGNGNTYLE (SEQ ID NO: 17), KSSQSLLNSGDQKNYLT (SEQ ID NO: 23) and RSSKSLLHSNGITYF (SEQ ID NO: 29); the CDR-L2 sequence may be any one of KVSNRFS (SEQ ID NO: 18), WASTRES (SEQ ID NO: 24) and QMSNLAS (SEQ ID NO: 30); and the CDR-L3 sequence may be any one of FQGSHVPYT (SEQ ID NO: 19), QNDYSYPLT (SEQ ID NO: 25) and AQNLELPPT (SEQ ID NO: 31). Specifically, the VL MUC1 It may comprise: (1) CDR-L1 sequence RSSQDIVYGNGNTYLE (SEQ ID NO: 17), CDR-L2 sequence KVSNRFS (SEQ ID NO: 18), and CDR-L3 sequence FQGSHVPYT (SEQ ID NO: 19); or (2) CDR-L1 sequence KSSQSLLNSGDQKNYLT (SEQ ID NO: 23), CDR-L2 sequence WASTRES (SEQ ID NO: 24), and CDR-L3 sequence QNDYSYPLT (SEQ ID NO: 25); or (3) CDR-L1 sequence RSSKSLLHSNGITYF (SEQ ID NO: 29), CDR-L2 sequence QMSNLAS (SEQ ID NO: 30), and CDR-L3 sequence AQNLELPPT (SEQ ID NO: 31). For example, the VL MUC1 It may comprise any of the following amino acid sequences:

[0168] (a) DVVMTQSPLSLPVTLGQPASISCRSSQDIVYGNGNTYLEWYLQRPGQSPRLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIK (SEQ ID NO: 9);

[0169] and

[0170] (c) DIVMTQSPLSNPVTPGEPASISCRSSKSLLHSNGITYFFWYLQKPGQSPQLLIYQMS NLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGQGTKVEIKR (SEQ ID NO: 13).

[0171] Alternatively, the VL MUC1 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 9, 11, and 13, and the differences in the amino acid sequences are all in the non-CDR regions.

[0172] In the above embodiments, the CL of polypeptide chain I and / or II MUC1 The domain may comprise the amino acid sequence shown below:

[0173] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 87).

[0174] According to the above embodiment, the polypeptide chain I and the polypeptide chain II may each comprise the amino acid sequence shown below:

[0175] DIVMTQSPLSNPVTPGEPASISCRSSKSLLHSNGITYFFWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGQGTKVEIKRRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:88).

[0176] In the above embodiment, the VH MUC1 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence. The CDR-H1 sequence may be any one of NYWMH (SEQ ID NO: 14), DHAIH (SEQ ID NO: 20) and NYWMN (SEQ ID NO: 26); the CDR-H2 sequence may be any one of YINPSSGYTQYNQKFKD (SEQ ID NO: 15), HFSPGNTDIKYNDKFKG (SEQ ID NO: 21) and EIRLKSNNYTTHYAESVKG (SEQ ID NO: 27); and the CDR-H3 sequence may be any one of YYGDYLFPY (SEQ ID NO: 16), STFFFDY (SEQ ID NO: 22) and HYYFDY (SEQ ID NO: 28). Specifically, the VH MUC1 It may comprise: (1) CDR-H1 sequence NYWMH (SEQ ID NO: 14), CDR-H2 sequence YINPSSGYTQYNQKFKD (SEQ ID NO: 15), and CDR-H3 sequence YYGDYLFPY (SEQ ID NO: 16); or (2) CDR-H1 sequence DHAIH (SEQ ID NO: 20), CDR-H2 sequence HFSPGNTDIKYNDKFKG (SEQ ID NO: 21), and CDR-H3 sequence STFFFDY (SEQ ID NO: 22); or (3) CDR-H1 sequence NYWMN (SEQ ID NO: 26), CDR-H2 sequence EIRLKSNNYTTHYAESVKG (SEQ ID NO: 27), and CDR-H3 sequence HYYFDY (SEQ ID NO: 28). For example, the VH MUC1 It may comprise any of the following amino acid sequences:

[0177] (a) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWMHWVRQAPGQGLEWMGYINPSSGYTQYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCSTYYGDYLFPYWGQGTTVTVSS (SEQ ID NO: 8);

[0178] (b) EVQLVQSGAEVKKPGESLKISCKISGYIFTDHAIHWVRQMPGKGLEVVMGHFSPGNTDIKYNDKFKGQVTLSVDRSINTAYLQWSSLKASDTAIYFCKTSTFFFDYWGQGTRVTVSS (SEQ ID NO: 10); and

[0179] (c) EVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMNWVRQAPGKGLEWVGEIRLKSNNYTTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFDYWGQGTLVTVSS (SEQ ID NO: 12).

[0180] Alternatively, the VH MUC1 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 8, 10, and 12, and the differences in the amino acid sequences are all in the non-CDR regions.

[0181] In the above embodiments, the exemplary (scFv) CD3 It may comprise any one of the following amino acid sequences:

[0182]

[0183]

[0184] Alternatively, in certain embodiments, the (scFv) CD3It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences shown in the table above, and the differences in the amino acid sequences are all in the non-CDR regions.

[0185] In the above embodiments, the exemplary (scFv) CD28 It may comprise any one of the following amino acid sequences:

[0186]

[0187] Alternatively, in certain embodiments, the (scFv) CD28 It may comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences shown in the table above, and the differences in the amino acid sequences are all in the non-CDR regions.

[0188] In the above embodiment, the polypeptide chain III comprises any one of the following amino acid sequences:

[0189] EVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMNWVRQAPGKGLEWVGEIRLKSNNYTTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRKKLTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(SEQ ID NO:89);Or EVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMNWVRQAPGKGLEWVGEIRLKSNNYTTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRKKLTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAFRGLIGGTNKKASGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTVSS(SEQ ID NO:90);

[0190] In the above embodiment, the polypeptide chain IV comprises the following amino acid sequence: (SEQ ID NO: 91).

[0191] As described above, in the above situation, the polypeptide chain I and the polypeptide chain III, the polypeptide chain II and the polypeptide chain IV of the multispecific antigen-binding protein together form one or more first binding domains capable of specifically binding to MUC1; further, the polypeptide chain III is connected to the C-terminus of its CH3 domain (scFv) CD3 To specifically bind to the CD3 antigen binding site, and the polypeptide chain IV is connected to the C-terminus of its CH3 domain (scFv) CD28to specifically bind to the CD28 antigen-binding site. In some embodiments, the polypeptide chain III and the polypeptide chain IV form a heterodimeric structure through the interaction of partial domains. For example, the CH3 domain portions of the polypeptide chain III and the polypeptide chain IV can each contain amino acid substitutions, and the amino acid substitutions in the CH3 domains of the polypeptide chain III and the polypeptide chain IV can enable the two CH3 domains to form a heterodimerization effect. More specifically, the CH3 domain can be altered using the "knob-in-hole" technique (also known as Knob-in-Hole or KiH), which is described in detail in several examples, such as International Publication No. WO 96 / 027011, Ridgway et al., 1996, Protein Eng. 9:617-21, and Merchant et al., 1998, Nat. Biotechnol. 16:677-81. It is mentioned that the interaction surface of the two CH3 domains is altered to increase the heterodimerization of the two heavy chains containing the two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "knob" and the other a "hole." The introduction of a disulfide bridge can further stabilize the heterodimer (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270:26-35) and improve yield. WO96 / 027011, in its embodiments, proposes mutations that can produce heterodimerization of the two polypeptide chains, for example, where the "knob" is located in the antibody constant region of one polypeptide chain, specifically the CH3 region; and the "hole" is located in the antibody constant region of the other polypeptide chain, specifically the CH3 region. More specifically, in one embodiment, a "knob" is constructed by replacing an amino acid residue at a specific position in the CH3 or CH3 domain interface of one polypeptide chain with an amino acid having a larger side chain volume, thereby generating a protrusion-like structure; non-limiting examples of such amino acid mutations or substitutions include S354C and / or T366W; and a "hole" is constructed by replacing an amino acid residue at a specific position in the CH3 or CH3 domain interface of the other polypeptide chain with an amino acid having a smaller side chain volume, thereby generating a cavity-like structure; non-limiting examples of such amino acid mutations or substitutions include Y349C, T366S, L368A, and / or Y407V. In these cases, the surface interaction between the two polypeptide chains is achieved through the chimerization of the "knob" and the "hole" to form a heterodimer.In addition, US10011858B2 and WO2009 / 089004 propose in their embodiments a "charge reversal strategy" in the heavy chain CH3 domain to support favorable electrostatic attractive interactions and promote the formation of the desired Fc heterodimer; for example, the polypeptide chain containing the "knob" mutation may further or alternatively include replacing the original amino acid residue with a negatively charged amino acid, and non-limiting examples of such amino acid mutations or substitutions are K370E and / or K439E; and the polypeptide chain containing the "hole" mutation may further or alternatively include replacing the original amino acid residue with a positively charged amino acid, and non-limiting examples of such amino acid mutations or substitutions are D356K and / or E357K.

[0192] In some other embodiments, the two polypeptide chains forming the heterodimer may further comprise other amino acid mutations or substitutions, including those that reduce the ADCC effect (antibody-dependent cell-mediated cytotoxicity, or antibody-dependent cell-mediated Common engineering schemes for reducing ADCC effects may include: (1) amino acid mutations, for example, L235A, G237A, and E318A (AAA mutations), L234A and L235A (LALA mutations), or L234A, L235A, and P329G (LALA-PG mutations); (2) glycosylation modification, for example, deglycosylated N297A, N297Q, or N297G (NA mutations); or (3) IgG4 / IgG2 antibody modification, for example, S228P and L235E (IgG4-PE mutations), or V234A, G237A, P238S, H268A, V309L, A330S, and P331S (IgG2c4d mutations).

[0193] The protruding "knob" structure, the cavity-like "hole" structure, the oppositely charged amino acid mutations or substitutions, and the engineered Fc modifications for reducing ADCC effects provided herein can all be created using synthetic methods, such as altering the nucleic acid encoding the polypeptide or through peptide synthesis. For example, the nucleic acids encoding the two polypeptide chains forming the heterodimer can be present in the same vector or in different vectors.

[0194] In some embodiments, the multispecific antigen-binding protein described herein comprises a "knob" mutation on polypeptide chain III and a "hole" mutation on polypeptide chain IV to form an IgG-like antibody structure. In some embodiments, the multispecific antigen-binding protein comprises a "knob" mutation on polypeptide chain IV and a "hole" mutation on polypeptide chain III to form an IgG-like antibody structure. In the above case, an exemplary polypeptide chain comprising a "knob" mutation may comprise the following features: amino acid substitutions at positions 297, 354, 366, 370, and 439 corresponding to human IgG1 according to the EU Index, wherein: the amino acid substitution at position 297 is N297A, the amino acid substitution at position 354 is S354C, the amino acid substitution at position 366 is T366W, the amino acid substitution at position 370 is K370E, and the amino acid substitution at position 439 is K439E. Also in the above situation, an exemplary polypeptide chain comprising a "hole" mutation may comprise the following features: according to the EU Index, it comprises amino acid substitutions at positions corresponding to human IgG1 at positions 297, 349, 356, 357, 366, 368 and 407, wherein: the amino acid substitution at position 297 is N297A, the amino acid substitution at position 349 is Y349C, the amino acid substitution at position 356 is D356K, the amino acid substitution at position 357 is E357K, the amino acid substitution at position 366 is T366S, the amino acid substitution at position 368 is L368A, and the amino acid substitution at position 407 is Y407V.

[0195] In summary of the above embodiments, the multispecific antigen-binding protein described in the present application may comprise any of the following amino acid sequence combinations:

[0196]

[0197]

[0198]

[0199] In another aspect, the present application provides a nucleic acid molecule encoding the antigen-binding protein described herein. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid can be used to treat or alleviate MUC1-related tumors or cancers. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to produce more stable mRNA. Known mRNA modification techniques can be roughly divided into three categories: synthesizing mRNA using artificially synthesized non-natural RNA instead of natural RNA; adding 5' caps, 3' poly (A) "tails," and UTR (untranslated region) sequences; and using special novel formulation technologies to effectively protect mRNA. Among these, the preferred mRNA modification technique is to synthesize mRNA using artificially synthesized non-natural RNA instead of natural RNA. Chemical modifications on eukaryotic mRNA can be roughly divided into three categories: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification can be selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0200] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an antigen-binding protein as described herein, which comprises a nucleotide sequence selected from any one of SEQ ID NOs: 92-107. In some embodiments, the nucleic acid molecule comprises: a nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II as described herein, and a nucleotide sequence encoding polypeptide chain III and polypeptide chain IV as described herein; wherein: (1) the nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is as shown in SEQ ID NO: 109, the nucleotide sequence encoding polypeptide chain III is as shown in SEQ ID NO: 110, and the nucleotide sequence encoding polypeptide chain IV is as shown in SEQ ID NO: 112; or (2) the nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is as shown in SEQ ID NO: 109, the nucleotide sequence encoding polypeptide chain III is as shown in SEQ ID NO: 111, and the nucleotide sequence encoding polypeptide chain IV is as shown in SEQ ID NO: 112.

[0201] On the other hand, the present application provides a vector comprising the nucleic acid molecules described herein. For example, the vector is a recombinant vector. Specifically, the recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. Recombinant vectors may include single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA or both; and other types of polynucleotides known in the art. For example, viral vectors can be used. Viral vectors may comprise virally derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV). Viruses and viral vectors can be used for in vitro, ex vivo and / or in vivo delivery.

[0202] For another example, the carrier is a delivery vector. In certain embodiments, the delivery vector comprises antigen-binding proteins as described herein, nucleic acid as described herein and / or recombinant vector as described herein, and optionally comprises liposome and / or lipid nanoparticle (LNP). For example, the delivery vector can be introduced into cells by physical delivery methods. The example of physical method comprises microinjection, electroporation and hydrodynamic delivery. For example, nucleic acid as described herein can be wrapped in cationic lipid particles (such as liposomes) by LNPs, and can be delivered to cells relatively easily. In some examples, lipid nanoparticle does not contain any viral components, which helps to minimize safety and immunogenicity problems. Lipid particle can be used for external, ex vivo and in vivo delivery. For example, the composition of LNP can comprise cationic lipid, ionizable lipid, pegylated lipid and / or support lipid, and optional cholesterol component.

[0203] On the other hand, the present application provides a cell comprising the antigen binding protein described herein, the nucleic acid molecule described herein, and / or the vector described herein.

[0204] On the other hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein described herein and a pharmaceutically acceptable carrier.

[0205] In another aspect, the present application provides use of the antigen binding protein described herein in the preparation of a medicament for treating MUC1-related tumors or cancers.

[0206] On the other hand, the present application provides a method for treating MUC1-expressing positive tumors or cancers, the method comprising administering to a patient in need thereof an effective amount of the antigen binding protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the pharmaceutical composition described herein. For example, the method of the present application may be an in vitro or ex vivo method. For example, the method of the present application may be a method for therapeutic purposes. For example, the method of the present application may be an in vivo method. For example, the method of the present application can achieve a tumor inhibition rate of about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or about 70% for the MUC1-expressing positive tumor of the patient compared to a patient who has not been administered the method of the present application. The patient's tumor tissue infiltrates human CD45 + Immune cells and CD3 + The proportion of T cells is increased by about 10%, about 20%, about 30%, about 40%, about 50%, or more compared to a patient not administered the method of the present application. For example, the MUC1-positive tumor can be a human pancreatic cancer BXPC-3 tumor, or can be a human breast cancer HCC1954 tumor. For example, the method comprises administering an effective amount of the antigen-binding protein described herein to the patient for about 2 to about 4 weeks, with the frequency of administration being two or more times per week. For example, the dose or concentration of the antigen-binding protein described herein administered in the method can exhibit a positive correlation with the tumor inhibition rate.

[0207] In some embodiments, the method further includes administering to the patient an effective amount of the antigen binding proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein, and / or the pharmaceutical compositions described herein, while using other anti-tumor drugs and / or anti-tumor treatments. For example, the other anti-tumor drugs include but are not limited to antibody drugs and / or small molecule chemotherapy drugs. For example, the anti-tumor treatment includes but is not limited to immune checkpoint blockade (ICB) therapy, chimeric antigen receptor (CAR)-T therapy, and / or oncolytic virus (OV) therapy. For example, simultaneous use can refer to the administration of two drugs or drugs of the present application and other treatment methods simultaneously, or successively in any order. For example, it may be preferred that two (or all) active ingredients of the drug simultaneously exert their biological activities for a certain period of time. For example, the drug of the present application and other drugs used simultaneously are not necessarily administered by the same route of administration. In the examples provided herein, the method further includes administering to the patient an effective amount of the antigen binding proteins described herein while using gemcitabine. For example, the method can produce about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 93% or about 98% tumor inhibition rate for human pancreatic cancer HPAC tumors, and the inhibition rate is compared to a control group of a vehicle combined with gemcitabine under the same administration conditions. For example, the method comprises administering an effective amount of the antigen binding protein described herein to the patient for about 2 weeks to about 4 weeks, the frequency of administration can be 2 times or more per week, and gemcitabine is used simultaneously during the period. For example, the method can alleviate or avoid the occurrence of human immune cells (such as CD45 + Immune cells and CD3 + In the case where the proportion of T cells is decreased, the other antibodies may refer to bispecific antibodies or antibodies that activate T cells based on a single CD3 signal.

[0208] In another aspect, the present application provides the antigen binding protein, nucleic acid molecule, vector, cell, and / or pharmaceutical composition described herein, for use in treating MUC1-positive tumors or cancers.

[0209] In some embodiments, the MUCl expression-positive tumor or cancer is selected from pancreatic cancer, ovarian cancer, breast cancer, lung adenocarcinoma, fallopian tube cancer, and colorectal cancer.

[0210] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0211] Example

[0212] Example 1

[0213] Construction, expression and purification of the eukaryotic expression vector of the trispecific antigen-binding protein of the present application

[0214] This example uses a mammalian cell expression system for antibody expression. A recombinant plasmid containing the nucleotide sequence encoding the MUC1×CD3×CD28 trispecific antibody described herein was transiently transferred into the expi293 (Gibco) suspension cell line via PEI (Polysciences)-mediated transfection. Transient expression was performed using standard culture procedures, and the cell culture supernatant was harvested by centrifugation.

[0215] The harvested supernatant was purified by affinity method. The target protein was captured using Capto L filler (purchased from Cytiva). First, the chromatography column was treated with affinity cleaning solution for 3-4 column volumes. Equilibrate at least 4 column volumes with affinity balancing solution. The clarified cell harvested solution was loaded once. After loading, equilibrate 3-4 column volumes with affinity balancing solution. Then rinse at least 4 column volumes with affinity rinsing solution. After rinsing, the protein was eluted with affinity elution solution. When the UV absorbance value rose to 10-50mAU / 2mm, the protein was collected. When the UV absorbance value dropped to 10-50mAU / 2mm, the collection was terminated. The eluted protein was stirred and weighed, and the protein concentration was determined by OD280 absorption method. The protein captured by Capto L affinity chromatography was then subjected to cation exchange chromatography ( -50SP, purchased from Nano-Tech) to obtain the purified protein.

[0216] Example 2

[0217] In vitro cell binding assay to evaluate the binding activity of the trispecific antigen binding protein of the present application to membrane-bound antigens

[0218] In this example, the binding activity of different MUC1×CD3×CD28 trispecific antigen binding proteins (such as the amino acid sequences shown in SEQ ID NOs: 71-73 and 78-81) to the membrane-bound antigens hMUC1, hCD3, and hCD28 was evaluated using an in vitro cell binding assay.

[0219] 1×10 per well 5 Target antigen-expressing cells (OVCAR3 and CAOV3, H9 or CHO-CD28) were added to a 96-well plate at a density of 10 cells / well. Negative cells that do not express the target protein were used as controls. Specifically, the cells and negative controls used for each antigen are as follows.

[0220] Detection of binding activity with hMUC1:OVCAR3 and CAOV3 cell lines were used, which express hMUC1 and have the hMUC1 phenotype + hCD3 - hCD28 - ; and the CHO-S cell line that does not express hMUC1, hCD3 and hCD28 was used as a negative control.

[0221] Detection of binding activity to hCD3 H9 cell line (purchased from CCTCC, GDC0031) was used, which is a human T lymphoma cell expressing hCD3 and has the phenotype of hMUC1. - hCD3 + hCD28 - ; and the CHO-S cell line that does not express hMUC1, hCD3 and hCD28 was used as a negative control.

[0222] Detection of binding activity to hCD28 :The CHO-CD28 cell line is a stable CHO cell line expressing hCD28, with a phenotype of hMUC1 - hCD3 - hCD28 + ; and the CHO-S cell line that does not express hMUC1, hCD3 and hCD28 was used as a negative control.

[0223] The MUC1×CD3×CD28 trispecific antigen-binding protein, purified according to the procedure described in Example 1, was diluted in STB staining buffer (PBS containing 1% vol FBS) and added to the 96-well plate to final concentrations of 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, 0.156 μg / mL, 0.039 μg / mL, and 0.01 μg / mL, respectively. The protein was incubated with the assay cells at 4°C for 1 hour. Unbound antibody was removed by centrifugation. Cells binding to the trispecific antigen-binding protein were detected using Biotin-Protein L (GenScript, Cat#: M00097) and APC-Streptavidin (Biolegend, Cat#: 405207), and mean fluorescence intensity (MFI) was measured using a Thermo Fisher Attune N×T flow cytometer.

[0224] The results are as follows Figures 2A-2E As shown, the binding of the trispecific antigen binding protein of the present application to the membrane-bound antigen hMUC1 presents a dose-dependent effect; the binding of the trispecific antigen binding protein to the antigen hCD28 EC 50 The value range is about 2.137-5.229μg / mL; binding to antigen hCD3 EC 50The values ​​ranged from approximately 2.386 to 8.318 μg / mL. The above results also showed that the MUC1×CD3×CD28 trispecific antigen binding protein molecule had no detectable binding activity with the negative cell line CHO-S, which does not express the target protein, thus demonstrating good target binding specificity.

[0225] Example 3

[0226] PBMC system in vitro evaluation of effector cell activation mediated by the trispecific antigen binding protein of the present application

[0227] This example uses human PBMCs as an evaluation system to test the ability of the MUC1×CD3×CD28 trispecific antigen-binding protein (amino acid sequences set forth in SEQ ID NOs: 71-73 and 78-81) to mediate effector cell activation in vitro. Specifically, different concentrations of the trispecific antigen-binding protein were co-incubated with PBMCs and target cells, and the expression of T cell activation markers CD25, CD69, and Granzyme B in the PBMCs was measured to assess the T cell activation effect of the trispecific antigen-binding protein.

[0228] Specifically, PBMC and target cells OVCAR3 were cultured at 1×10 5 pcs / well and 1×10 4 The density of cells / well was added to a 96-well flat-bottom cell culture plate. The MUC1×CD3×CD28 trispecific antigen binding protein obtained according to the purification process of Example 1 was serially diluted: the final concentration was set to 0.97ng / mL, 3.9ng / mL, 15.6ng / mL, 62.5ng / mL, 250ng / mL, and 1000ng / mL, and the vehicle control group of RPMI 1640 complete medium was used as a negative control. The serially diluted antigen binding proteins were added to 96-well flat-bottom cell culture plates and incubated with PBMCs at 37°C and 5% CO2 for 24 hours. After washing the plates, the cells were stained with detection antibodies. The following detection antibodies were used:

[0229] Detection of antibodies factory batch number APC-Cy7 anti-human CD4 antibody BioLegend 300518 FITC anti-human CD8 antibody BD Bioscience 555634 APC anti-human CD25 antibody BioLegend 356110 PE anti-human CD69 antibody BioLegend 310906 BV421 anti-human / mouse Granzyme B antibody BioLegend 396414

[0230] The ratio of the following target cell subsets in each well of the 96-well plate was then detected using a Thermo Fisher Attune N×T flow cytometer: CD4 + CD25 + 、CD4 + CD69 + 、CD4 + Granzyme B + 、CD8 + CD25 + 、CD8 +CD69 + and CD8 + Granzyme B + cell.

[0231] The results are as follows Figures 3A-3F The results showed that compared with the vehicle control, the trispecific antigen molecules of the present application (shown as SEQ60-1, SEQ60-2 and SEQ58-2, whose amino acid sequences are shown in SEQ ID NOs: 78, 79 and 72, respectively) expressed CD25 β-catenin after 24 hours of incubation with PBMC. + 、CD69 + and Granzyme B + CD4 + and CD8 + The cell proportions were significantly increased and showed a dose-related relationship with the concentration of antigen-binding protein, demonstrating good effector cell activation ability.

[0232] Example 4

[0233] Jurkat-NFAT system to evaluate T cell activation mediated by the trispecific antigen binding protein of the present application in vitro effect

[0234] This example uses Jurkat-NFAT as an evaluation system to test the ability of different specific antigen binding proteins to mediate effector cell activation as shown in the table below. Specifically, different concentrations of the above-mentioned antigen binding protein to be tested are incubated with Jurkat-NFAT and target cells, human papillary ovarian adenocarcinoma cells CAOV3, human lung cancer adenocarcinoma cells DV90, and human breast cancer cells HCC1937, respectively. The expression of luciferase driven by the NFAT response element (NFAT-RE) after activation of the Jurkat-NTAT cell line is detected, and the bioluminescent signal is detected and quantified by adding a luciferase substrate, thereby observing the activation of T cells by the antigen binding protein to be tested.

[0235]

[0236] Note: (1) The spatial configuration of the SAR-SEQ60 molecule in the control group is as follows Figure 4D As shown (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).). (2) null indicates that the SEQ60-2-2C molecule of the other control group (scFv)CD3 The domains were replaced with non-functional antibody sequences.

[0237] The specific process includes: (1) taking target cells and preparing 1×10 5 After the cell suspension was prepared into 100 μL per well, it was inoculated into a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 24 h to adhere to the wall; (2) The stable strain of Jurkat-NFAT cells (Novoprotein) was taken and prepared with RPMI 1640 + 0.5% BSA to 2.5×10 5 After preparing a cell suspension of 10 cells / mL, 80 μL was inoculated into a 96-well cell culture plate; (3) 20 μL of the test solution was added to each well in a gradient dilution; (4) the mixture was mixed and incubated at 37°C, 5% CO2 for 6 h; (5) 30 μL of KeyTec Ultra Luciferase Detection reagent (VEKY-BIO) was added to each well, mixed and the fluorescence value (RLU) of each well was read using a microplate reader.

[0238] The results are as follows Figures 4A-4C The results showed that in the presence of three different target cells, CAOV3, DV90, and HCC1937, trispecific antibodies with different configurations of MUC1×CD3×CD28 were able to induce varying degrees of effector cell activation, and all exhibited a clear dose-dependent effect. Among them, the trispecific antigen-binding protein with a tandem scFv structure (shown as SEQ60-2, whose amino acid sequence is shown in SEQ ID NO:79) had the best activation effect on Jurkat cells, and was superior to the drug combination of MUC1×CD3 bispecific antigen-binding protein and CD28 scFv in activating Jurkat cells.

[0239] Example 5

[0240] In vitro evaluation of the killing activity of human PBMCs on target cells mediated by the trispecific antigen binding protein of the present application

[0241] This example evaluates the cytotoxicity of human PBMCs against target cells mediated by the constructed antigen-binding proteins of different specificities (as listed in Example 4). Human PBMCs isolated from healthy volunteers were used as effector cells, and human papillary ovarian adenocarcinoma cells (CAOV3), human lung adenocarcinoma cells (DV90), human pancreatic acinar epithelial carcinoma (HPAC), and human breast cancer cells (HCC1937) were used as target cells. After incubation of the PBMCs and target cells with different concentrations of the test articles, the number of target cells in the system was detected. The cytotoxicity of human PBMCs against the different target cells mediated by the MUC1×CD3×CD28 trispecific antigen-binding protein was evaluated.

[0242] Specifically, PBMCs from 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 a 96-well flat-bottom cell culture plate at a ratio of 10:1 (PBMC: 2×10 5 Target cells: 2×10 4 The test sample was serially diluted in RPIM 1640 complete medium: the final concentration of the antigen-binding protein to be tested was set to 0, 0.00768nM, 0.0384nM, 0.192nM, 0.96nM, 4.8nM, 24nM, and 120nM, and then added to the above-mentioned 96-well flat-bottom cell culture plate. After incubation at 37°C, 5% CO2 for 120 hours, the plate was washed and 100μL of RPIM 1640 complete medium containing 10% CCK8 stock solution was added. The reaction was continued at 37°C for 1-4 hours, and the OD450 was measured using a microplate reader. The cell killing efficiency was calculated according to the following formula; two replicates were set for each group, and a vehicle control with RPMI 1640 complete medium was used as a blank control. The killing efficiency was calculated according to the following formula:

[0243]

[0244] The data were analyzed using GraphPad Prism software. The antibody-mediated cytotoxicity of human PBMC against target cells was expressed as 50% maximal effect concentration (EC). 50 Value representation.

[0245] The results are as follows Figures 5A-5D The results showed that compared with the bispecific antigen binding protein, the effector cells mediated by the MUC1×CD3×CD28 trispecific antigen binding protein had stronger killing activity against various tumor cell lines, especially the trispecific antigen binding protein with a tandem scFv structure (shown as SEQ60-2, whose amino acid sequence is shown in SEQ ID NO:79) had the strongest killing activity against various tumor cell lines tested; the EC killing activity of human PBMCs mediated by it against CAOV3, DV90, HPAC, and HCC1937 was stronger than that of the other two proteins. 50 The values ​​were 0.07966nM, 0.02002nM, 0.03046nM, and 0.4317nM, respectively. The EC values ​​of human PBMCs mediated by MUC1×CD3 bispecific antigen binding protein (SEQT60-C1) against CAOV3, DV90, and HPAC were 50 The values ​​were 0.5657nM, 59.68nM, and 19.63nM, respectively; no normal EC was fitted for HCC1937. 50 value.

[0246] Example 6

[0247] In vitro evaluation of effector cell proliferation mediated by the trispecific antigen binding protein of the present application

[0248] This example evaluated the T cell proliferation ability mediated by the MUC1×CD3×CD28 trispecific antigen binding protein (SEQ60-1 and SEQ60-2 shown in Example 4), and compared it with the treatment group treated with the MUC1×CD3 bispecific antigen binding protein in combination with CD28 scFv (SEQT60-C1+CD28 scFv shown in Example 4). Human PBMCs isolated from healthy volunteers were used as the evaluation system. After incubating the PBMCs and target cells with different concentrations of the test antibody, the CD4 + CellTrace cFSE dim , and CD8 + CellTrace cFSE dim cell population, thereby examining the T cell proliferation induced by the antigen binding protein to be tested.

[0249] Specifically, PBMCs from healthy volunteers were extracted with Ficoll-Paque Plus (GE, Cat#: 17-1440-02) and incubated with 5 μM tracking dye (eBioscience CFSE) at 37°C for 20 min. 5 The cells were added to a 96-well flat-bottom cell culture plate at a density of 100 μg / well. The antigen-binding proteins to be tested obtained according to the purification process of Example 1 were serially diluted with RPMI 1640 complete medium: the final concentrations of the antigen-binding proteins were set to 0, 18 μM, 55 μM, and 168 μM. A vehicle control group of RPMI 1640 complete medium was used as a negative control. The diluted antigen-binding proteins were added to the above-mentioned 96-well flat-bottom cell culture plate, and target cells DV90 were added at a ratio of 10:1 between target cells and the cells. After incubation at 37°C and 5% CO2 for 96 hours, the cells were stained with APC / Cyanine7 anti-human CD4 antibody (BioLegend, Cat#:300518) and FITC anti-human CD8 antibody (BD Bioscience, Cat#:555634), and the CD4 in PBMCs in each well was detected using a Thermo Fisher Attune N×T flow cytometer. + CellTrace cFSE dim 、CD8 + CellTrace cFSE dim Cell clusters.

[0250] The results are as follows Figure 6As shown, the MUC1×CD3×CD28 trispecific antigen binding proteins SEQ60-1 and SEQ60-2 antibodies can induce strong CD4 + T and CD8 + Compared with the trispecific antigen binding protein, the MUC1×CD3 bispecific antigen binding protein SEQT60-C1 combined with anti-CD28 scFv had a weaker effect on T cell proliferation and could not induce CD4 T cell proliferation under all tested concentrations. + T cell proliferation, CD8 + The proliferation of T cells also only appeared in the high-dose group, and the proliferation effect was very weak.

[0251] Example 7

[0252] Effects of the trispecific antigen binding protein of the present application on patient-derived tumor organoids

[0253] Organoids are 3D cell cultures grown in vitro using the self-assembly properties of stem cells. They share highly similar histological, genetic, and physiological characteristics with real tissues and can serve as miniature replicas of real tissues and organs. In this example, organoid models constructed from tumor tissues from two different cancer patients (4-58-T lung cancer and 10-2-T ovarian cancer) were used to evaluate the pharmacodynamic effects of the MUC1×CD3×CD28 trispecific antibody and compared it with the pharmacodynamic effects of the MUC1×CD3 bispecific antibody. The specific antigen-binding proteins in each group are shown in the table below.

[0254]

[0255] 4-58-T lung cancer organoid model :

[0256] The specific process includes: (1) First, 1000 cells / well of organoids were plated in a 384-well plate; (2) After the organoids had settled stably to the bottom of the plate, 20,000 cells / well of T cells were added. The effector-target ratio was 20:1; (3) After the T cells had evenly settled to the bottom of the plate, different concentrations of antigen-binding protein were added in a gradient dilution, and the volume of each well was adjusted to 100 μL, with duplicate wells for each group. (4) After incubation for 139 hours at 37°C and 5% CO2, the experiment was terminated, bright field photos of the incubated cells were taken, and the supernatant was collected by centrifugation to detect LDH and cytokine release.

[0257] The results are as follows Figures 7A-7FThe results showed that compared with the control group (4-58-T lung cancer organoids + T cells), the MUC1×CD3×CD28 trispecific antigen binding proteins SEQ60-1, SEQ60-2, and SEQ60-5 significantly induced the proliferation of T cells; the end-point supernatant LDH data showed that the killing effect of the MUC1×CD3×CD28 trispecific antigen binding proteins with different configurations on tumor organoids was better than that of the MUC1×CD3 bispecific antibody ( Figure 8 The killing effect induced by the SEQ60-2 treatment group was the most obvious and concentration-dependent, followed by the SEQ60-1 treatment group. The above results are consistent with the results of cytokine release. The trispecific antigen binding proteins SEQ60-1 and SEQ60-2 induced strong TNFα and IFNγ cytokine release ( Figures 9A-9C ).

[0258] 10-2-T ovarian cancer organoid model :

[0259] The specific process includes: (1) First, 1000 cells / well of organoids were plated in a 384-well plate; (2) After the organoids had settled stably to the bottom of the plate, 20,000 cells / well of T cells were added. The effector-target ratio was 20:1; (3) After the T cells had evenly settled to the bottom of the plate, different concentrations of antigen-binding protein were added in a gradient dilution, and the volume of each well was adjusted to 100 μL, with duplicate wells for each group. (4) After incubation for 120 hours at 37°C and 5% CO2, the experiment was terminated, bright field photos of the incubated cells were taken, and the supernatant was collected by centrifugation to detect LDH and cytokine release.

[0260] The results are as follows Figures 10A-10D The results showed that compared with the control group (10-2-T ovarian cancer organoids + T cells), the trispecific antigen binding protein SEQ60-2 of MUC1×CD3×CD28 significantly induced the proliferation of T cells; the end-point supernatant LDH data showed that the killing effect of SEQ60-2 with different configurations on tumor organoids was better than that of the MUC1×CD3 bispecific antigen binding protein SEQT60-C1 and the combination of SEQT60-C1+anti-CD28 scFv ( Figure 11 The above results are consistent with the results of cytokine release. The trispecific antigen binding protein SEQ60-2 induced strong IL2 and IFNγ cytokine release ( Figures 12A-12B ).

[0261] Example 8

[0262] The tumor-suppressing effect of the trispecific antigen binding protein of the present application in a subcutaneous transplanted tumor model

[0263] Human pancreatic cancer BXPC-3 tumor model implanted subcutaneously in hPBMC-NPG mice :

[0264] This example evaluated the tumor inhibitory effect of the MUC1×CD3×CD28 trispecific antigen binding protein in a hPBMC humanized NPG mouse subcutaneously transplanted human pancreatic cancer BXPC-3 tumor model. Specifically, 6-8 week old female immunodeficient NPG mice (Beijing Weitongda Animal Breeding Co., Ltd.) were subcutaneously inoculated with 1×10 7 BXPC-3 cells were seeded in a volume of 200 μL. Animal operation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pengli Biotechnology.

[0265] When the tumor grows to about 63 mm 2 At the same time, 20 tumor-bearing mice were randomly divided into 5 groups (4 mice / group) according to body weight and tumor volume, including vehicle control group (G1), 500 μg / kg and 150 μg / kg dose groups of SEQ60-1 (G2-G3), and 500 μg / kg and 150 μg / kg dose groups of SEQ60-2 (G4-G5). On the day of grouping, 1×10 7 PBMC (100 μL) were infused back through the tail vein. Each group of mice was intravenously administered twice a week for 4 weeks, for a total of 8 times. The body weight and tumor volume of the mice were measured and recorded twice a week, and the following indicators were calculated: tumor volume (V), tumor growth inhibition rate (TGI TV ), and inhibition rate of tumor weight (IRTW%).

[0266] The results are as follows Figures 13A-13C As shown, the MUC1×CD3×CD28 trispecific antigen binding proteins SEQ60-1 and SEQ60-2 significantly inhibited tumor growth at both 150 μg / kg and 500 μg / kg doses (P<0.01), and the reduction in tumor volume was dose-dependent with increasing concentration of the antigen binding protein. At the study endpoint, tumors were removed, weighed, and the inhibition rate of tumor weight (IRTW%) was calculated. The results showed that the IRTW% values ​​for groups G2-G5 at the study endpoint were 61.86%, 35.57%, 68.04%, and 53.09%, respectively.

[0267] Compared with the vehicle control (G1), there was no significant difference in the body weight of mice in the SEQ60-1 and SEQ60-2 SP administration groups (P>0.05).

[0268] huHSC NCG mouse subcutaneous transplantation of human breast cancer HCC1954 tumor model :

[0269] This example evaluated the tumor inhibitory effect of the MUC1×CD3×CD28 trispecific antigen binding protein in a human breast cancer HCC1954 tumor model in which humanized huHSC-derived NCG mice were subcutaneously transplanted. Specifically, female huHSC-NCG mice were subcutaneously inoculated with 5×10 6 HCC1954 cells. When the tumor grows to about 62 mm 2 At the time of the study, 20 tumor-bearing mice were randomly divided into five groups (4 mice / group) based on flow cytometry data of hCD45% × hCD3% cells in peripheral blood, mouse body weight, and tumor size. The groups included a vehicle control group (G1), SEQ60-1 500 μg / kg and 150 μg / kg dose groups (G2-G3), and SEQ60-2 500 μg / kg and 150 μg / kg dose groups (G4-G5). Animal operation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pengli Biological.

[0270] Each group of mice was intravenously administered twice a week for 4 weeks, for a total of 8 times. The body weight and tumor volume of the mice were measured and recorded twice a week, and the following indicators were calculated: tumor volume (V), tumor growth inhibition rate (TGI TV ), and inhibition rate of tumor weight (IRTW%).

[0271] The results are as follows Figures 14A-14C As shown, the MUC1×CD3×CD28 trispecific antigen binding proteins SEQ60-1 and SEQ60-2 significantly inhibited tumor growth at doses of 150 μg / kg and 500 μg / kg, with the reduction in tumor volume occurring in a dose-dependent manner with increasing antibody concentration. Tumors were harvested and weighed at the study endpoint, and the inhibition rate of tumor weight (IRTW%) was calculated. The results showed that the IRTW% values ​​for groups G2-G5 at the study endpoint were 60.81%, 39.63%, 55.94%, and 45.68%, respectively.

[0272] Human pancreatic cancer HPAC tumor model transplanted subcutaneously in hPBMC NOG mice :

[0273] This example evaluated the tumor inhibition effect of the MUC1×CD3×CD28 trispecific antigen binding protein in a human pancreatic cancer HPAC tumor model transplanted subcutaneously in hPBMC humanized NOG mice. In the experiment, the MUC1×CD3 bispecific antigen binding protein SEQT60-C1 was used as a control to compare the tumor inhibition effect of the trispecific antigen binding protein. Specifically, 1×10 7HPAC cells were seeded in a volume of 200 μL. Animal operation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Alamo Medical Technology Co., Ltd.

[0274] When the tumor grows to about 94 mm 2 At the same time, 20 tumor-bearing mice were randomly divided into 4 groups (5 mice / group) according to body weight and tumor volume, including vehicle control (G1), SEQ60-1 (G2), SEQ60-2 (G3), and SEQT60-C1 (G4). Each group of mice was intravenously administered twice a week for 5 weeks, for a total of 10 times. On the day of grouping, 5×10 6 PBMC (100 μL) was reinfused through the tail vein. In the first week, the dosage of each dosing group was 150 μg / kg. Starting from the second week, the dosage of G4 group was changed to 360 μg / kg, and the dosage of G2 and G3 groups remained unchanged. On D9 and D12 days after grouping, all groups of mice were given 30 mg / kg gemcitabine. The body weight and tumor volume of mice were measured and recorded every 2-3 days, and the tumor volume (V) and tumor growth inhibition rate (TGI) were calculated. At the end of the study, the tumor was removed, the tumor weight was weighed, and the tumor weight inhibition rate (IRTW%) was calculated. In addition, the collected tumor tissue samples were subjected to flow cytometry to analyze the T cell infiltration of the tumor tissue.

[0275] The results are as follows Figures 15A-15D As shown in the figure, the combination of MUC1×CD3×CD28 trispecific antigen binding protein SEQ60-2 and gemcitabine has a strong inhibitory effect on tumor growth. After the administration of 30mg / kg gemcitabine on D9 and D12, SEQ60-2 began to show a more obvious tumor inhibition effect. The tumor inhibition rate at the end of the study was TGI TV The % value reaches 93.01%.

[0276]

[0277] Flow cytometric analysis of changes in hCD45% × hCD3% revealed that, after four weeks of continuous administration, the proportion of tumor-infiltrating human immune cells as a percentage of all viable cells in the G4 bispecific antigen-binding protein SEQT60-C1 group was significantly reduced compared to the G1 vehicle control group at the study endpoint. However, the proportion of tumor-infiltrating human immune cells as a percentage of all viable cells in the trispecific antigen-binding protein SEQ60-1 and SEQ60-2 groups remained unchanged compared to the G1 vehicle control group. Analysis of tumor-infiltrating human T cells also revealed a significant decrease in T cell infiltration in the G4 group compared to the G1 vehicle control group at the four-week study endpoint, while no such decrease was observed in the trispecific antigen-binding protein-treated groups. This suggests that trispecific antigen-binding proteins offer a significant advantage in preventing cell death following T cell activation induced by a single CD3 signal.

Claims

1. A multispecific antigen-binding protein comprising (1) a first binding domain capable of specifically binding to human tumor-associated mucin-1 (TA-MUC1), wherein the first binding domain comprises a heavy chain variable region (VH MUC1 ) and light chain variable region (VL MUC1 ); (2) a second binding domain capable of specifically binding to a human CD3 polypeptide, wherein the second binding domain comprises a heavy chain variable region (VH CD3 ) and light chain variable region (VL CD3 );and (3) a third binding domain capable of specifically binding to a human CD28 polypeptide, wherein the third binding domain comprises a heavy chain variable region (VH CD28 ) and light chain variable region (VL CD28 ).

2. The antigen binding protein according to claim 1, wherein the first binding domain, the second binding domain and the third binding domain are single-chain antibody (scFv) fragments.

3. The antigen binding protein according to claim 1 or 2, wherein the first binding domain VH MUC1 The C-terminus of the first binding domain VL MUC1 The N-terminus of the first binding domain VL is connected via a first linker sequence, or the first binding domain VL MUC1 The C-terminus of the first binding domain VH MUC1 The N-terminus of the peptide is connected via a first linker sequence.

4. The antigen binding protein according to any one of claims 1 to 3, wherein the VH of the second binding domain is CD3 The C-terminus of the second binding domain VL CD3 The N-terminus of the second binding domain is connected via a first linker sequence, or the VL CD3 The C-terminus of the second binding domain VH CD3 The N-terminus of the peptide is connected via a first linker sequence.

5. The antigen binding protein according to any one of claims 1 to 4, wherein the VH of the third binding domain is CD28 The C-terminus of the third binding domain VL CD28 The N-terminus of the third binding domain is connected via a first linker sequence, or the VL CD28 The C-terminus of the third binding domain VH CD28 The N-terminus of the peptide is connected via a first linker sequence.

6. The antigen binding protein according to any one of claims 2 to 5, wherein the scFv fragment of the first binding domain is connected to the scFv fragment of the second binding domain via a first linker sequence.

7. The antigen binding protein according to any one of claims 2 to 6, wherein the scFv fragment of the second binding domain is connected to the scFv fragment of the third binding domain via a second linker sequence.

8. The antigen binding protein according to any one of claims 2-7, which comprises, in order from N-terminus to C-terminus: a scFv of a first binding domain, a first linker sequence, a scFv of a second binding domain, a second linker sequence and a scFv of a third binding domain.

9. The antigen binding protein according to any one of claims 2 to 8, wherein the connection between the scFv fragment of the first binding domain and the scFv fragment of the second binding domain is by any of the following methods: (1) VH of the first binding domain MUC1 The C-terminus of the second binding domain VH CD3 The N-terminus of (2) VL of the first binding domain MUC1 The C-terminus of the second binding domain VH CD3 The N-terminus of (3) VH of the first binding domain MUC1 The C-terminus of the second binding domain VL CD3 The N-terminus of (4) VL of the first binding domain MUC1 The C-terminus of the second binding domain VL CD3 The N-terminus of the peptide is connected via the first linker sequence.

10. The antigen binding protein according to any one of claims 2 to 9, wherein the connection between the scFv fragment of the second binding domain and the scFv fragment of the third binding domain is by any one of the following methods: (1) VH of the second binding domain CD3 The C-terminus of the third binding domain VH CD28 The N-terminus of (2) VL of the second binding domain CD3 The C-terminus of the third binding domain VH CD28 The N-terminus of (3) VH of the second binding domain CD3 The C-terminus of the third binding domain VL CD28 The N-terminus of (4) VL of the second binding domain CD3 The C-terminus of the third binding domain VL CD28 The N-terminus of the peptide is connected via the second linker sequence.

11. The antigen-binding protein according to any one of claims 2 to 10, comprising, in order from N-terminus to C-terminus: (1) VH of the first binding domain MUC1 , a first linker sequence, a VL of the first binding domain MUC1 , a first linker sequence, a VH of the second binding domain CD3 , a first linker sequence, the second binding domain VL CD3 , a second linker sequence, a VH of the third binding domain CD28 , a first linker sequence, a VL of the third binding domain CD28 ,or (2) VL of the first binding domain MUC1 , a first linker sequence, a VH of the first binding domain MUC1 , a first linker sequence, a VH of the second binding domain CD3 , a first linker sequence, the second binding domain VL CD3 , a second linker sequence, a VH of the third binding domain CD28 , a first linker sequence, a VL of the third binding domain CD28 .

12. The antigen binding protein according to any one of claims 1 to 11, wherein the VH MUC1 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein, The CDR-H1 sequence is shown in any one of SEQ ID NOs: 14, 20 and 26, the CDR-H2 sequence is shown in any one of SEQ ID NOs: 15, 21 and 27, and the CDR-H3 sequence is shown in any one of SEQ ID NOs: 16, 22 and 28.

13. The antigen binding protein according to any one of claims 1 to 12, wherein the VL MUC1 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein, The CDR-L1 sequence is shown in any one of SEQ ID NOs: 17, 23 and 29, the CDR-L2 sequence is shown in any one of SEQ ID NOs: 18, 24 and 30, and the CDR-L3 sequence is shown in any one of SEQ ID NOs: 19, 25 and 31.

14. The antigen binding protein according to any one of claims 1 to 13, wherein the VH MUC1 The amino acid sequence comprises any one of SEQ ID NOs: 8, 10 and 12, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 8, 10 and 12.

15. The antigen binding protein according to any one of claims 1 to 14, wherein the VL MUC1 The amino acid sequence comprises any one of SEQ ID NOs: 9, 11 and 13, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 9, 11 and 13.

16. The antigen binding protein according to any one of claims 1-15, wherein the scFv fragment of the first binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-37, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 32-37.

17. The antigen binding protein according to any one of claims 1 to 16, wherein the VH CD3 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein, The CDR-H1 sequence is shown as SEQ ID NO:42 or 48, the CDR-H2 sequence is shown as SEQ ID NO:43 or 49, and the CDR-H3 sequence is shown as SEQ ID NO:44 or 50.

18. The antigen binding protein according to any one of claims 1 to 17, wherein the VL CD3 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein, The CDR-L1 sequence is shown as SEQ ID NO:45 or 51, the CDR-L2 sequence is shown as SEQ ID NO:46 or 52, and the CDR-L3 sequence is shown as SEQ ID NO:47 or 53.

19. The antigen binding protein according to any one of claims 1 to 18, wherein the VH CD3 The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 38 or 40, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 38 or 40.

20. The antigen binding protein according to any one of claims 1 to 19, wherein the VL CD3 The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 39 or 41, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 39 or 41.

21. The antigen binding protein according to any one of claims 1-20, wherein the scFv fragment of the second binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 54-59, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 54-59.

22. The antigen binding protein according to any one of claims 1 to 21, wherein the VH CD28 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein, The CDR-H1 sequence is shown in SEQ ID NO:62, the CDR-H2 sequence is shown in SEQ ID NO:63, and the CDR-H3 sequence is shown in SEQ ID NO:

64.

23. The antigen-binding protein according to any one of claims 1 to 22, wherein the VL CD28 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein, The CDR-L1 sequence is shown in SEQ ID NO:65, the CDR-L2 sequence is shown in SEQ ID NO:66, and the CDR-L3 sequence is shown in SEQ ID NO:

67.

24. The antigen binding protein according to any one of claims 1 to 23, wherein the VH CD28 The amino acid sequence of SEQ ID NO: 60 is included, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO:

60.

25. The antigen-binding protein according to any one of claims 1 to 24, wherein the VL CD28 The amino acid sequence of SEQ ID NO:61 is included, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO:

61.

26. The antigen binding protein according to any one of claims 1-25, wherein the scFv fragment of the third binding domain comprises the amino acid sequence shown in SEQ ID NO: 68 or 69, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 68 or 69.

27. The antigen-binding protein according to any one of claims 3-6, 8, 9 and 11, wherein the first linker sequence comprises (G4S) n 、(G2S) n or (G2S) n GG, wherein n is an integer selected from 1 to 4.

28. The antigen-binding protein according to claim 27, wherein the first linker sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 1-4.

29. The antigen binding protein according to any one of claims 7, 8, 10 and 11, wherein the second linker sequence comprises an amino acid sequence of an IgD hinge region or a portion of an IgD hinge region.

30. The antigen-binding protein according to claim 29, wherein the second linker sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 5-7.

31. The antigen binding protein according to any one of claims 1-30, comprising an amino acid sequence selected from any one of SEQ ID NOs: 70-85.

32. The antigen-binding protein according to claim 1, comprising four polypeptide chains I-IV, wherein the polypeptide chains I-IV constitute an IgG-like antibody structure and each comprises a structure represented by the following formula (I) to (IV): (I) and (II) VL MUC1 -CL MUC1 ; (III) VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD3 ;as well as (IV)VH MUC1 -CH1-hinge region-CH2-CH3-(scFv) CD28 , in, The VL MUC1 and CL MUC1 They are the immunoglobulin light chain variable region and light chain constant region that specifically bind to MUC1, respectively.

33. The antigen binding protein of claim 32, wherein the VL MUC1 Comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence; wherein, The CDR-L1 sequence is SEQ ID NO:29, the CDR-L2 sequence is SEQ ID NO:30, and the CDR-L3 sequence is SEQ ID NO:

31.

34. The antigen-binding protein according to claim 32 or 33, wherein the VL MUC1 The amino acid sequence of SEQ ID NO: 13 is included, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO:

13.

35. The antibody according to any one of claims 32-34, wherein the CL of polypeptide chain I MUC1 The domain comprises the amino acid sequence shown in SEQ ID NO:

87.

36. The antigen-binding protein according to any one of claims 32-35, wherein each of the polypeptide chain I and the polypeptide chain II comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

88.

37. The antigen binding protein according to any one of claims 32-36, wherein the VH MUC1 Comprising a CDR-H1 sequence, a CDR-H2 sequence and a CDR-H3 sequence; wherein, The CDR-H1 sequence is shown in SEQ ID NO:26, the CDR-H2 sequence is shown in SEQ ID NO:27, and the CDR-H3 sequence is shown in SEQ ID NO:

28.

38. The antigen binding protein according to any one of claims 32-37, wherein the VH MUC1 The amino acid sequence of SEQ ID NO: 12 is included, or an amino acid sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO:

12.

39. The antigen binding protein according to any one of claims 32-38, wherein the (scFv) CD3 The fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 56 or 59, or comprises an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NO: 56 or 59.

40. The antigen binding protein according to any one of claims 32-39, wherein the (scFv) CD28 The fragment comprises the amino acid sequence shown in SEQ ID NO:69, or comprises an amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in SEQ ID NO:

69.

41. The antigen-binding protein according to any one of claims 32-40, wherein the polypeptide chain III comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89 or 90.

42. The antigen-binding protein according to any one of claims 32-41, wherein the polypeptide chain IV comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

91.

43. The antigen binding protein according to any one of claims 32-42, wherein the CH3 domain portions of the polypeptide chain III and the polypeptide chain IV respectively comprise amino acid substitutions, and the amino acid substitutions in the CH3 domains of the polypeptide chain III and the polypeptide chain IV enable the two CH3 domains to form heterodimerization.

44. The antigen binding protein according to any one of claims 32-43, wherein: (1) the CH3 domain of one of the polypeptide chain III and the polypeptide chain IV comprises amino acid substitutions at positions 297, 349, 356, 357, 366, 368 and 407 corresponding to human IgG1 according to the EU Index, wherein the amino acid substitution at position 297 is N297A, the amino acid substitution at position 349 is Y349C, the amino acid substitution at position 356 is D356K, the amino acid substitution at position 357 is E357K, the amino acid substitution at position 366 is T366S, the amino acid substitution at position 368 is L368A, and the amino acid substitution at position 407 is Y407V; and (2) the CH3 domain of the other of the polypeptide chain III and the polypeptide chain IV comprises amino acid substitutions at positions 297, 354, 366, 370 and 439 corresponding to human IgG1 according to the EU Index, the amino acid substitution at position 297 is N297A, the amino acid substitution at position 354 is S354C, the amino acid substitution at position 366 is T366W, the amino acid substitution at position 370 is K370E, and the amino acid substitution at position 439 is K439E.

45. The antigen-binding protein according to any one of claims 32-44, comprising one of the following amino acid sequence combinations: (1) polypeptide chains I and II respectively comprising the amino acid sequence of SEQ ID NO:88, polypeptide chain III comprising the amino acid sequence of SEQ ID NO:89, and polypeptide chain IV comprising the amino acid sequence of SEQ ID NO:91; or (2) polypeptide chains I and II respectively comprising the amino acid sequence of SEQ ID NO:88, polypeptide chain III comprising the amino acid sequence of SEQ ID NO:90, and polypeptide chain IV comprising the amino acid sequence of SEQ ID NO:

91.

46. ​​A nucleic acid molecule encoding the antigen binding protein of any one of claims 1-45.

47. The nucleic acid molecule of claim 46, comprising a nucleotide sequence encoding the antigen binding protein of any one of claims 1-31.

48. The nucleic acid molecule according to claim 47, comprising a nucleotide sequence selected from any one of SEQ ID NOs: 92-107.

49. The nucleic acid molecule according to claim 46, comprising: a nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II according to any one of claims 32 to 45, and a nucleotide sequence encoding polypeptide chain III according to any one of claims 32 to 45 and polypeptide chain IV; wherein: (1) the nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is shown in SEQ ID NO: 109, the nucleotide sequence encoding polypeptide chain III is shown in SEQ ID NO: 110, and the nucleotide sequence encoding polypeptide chain IV is shown in SEQ ID NO: 112; or (2) The nucleotide sequence encoding polypeptide chain I and / or polypeptide chain II is shown as SEQ ID NO:109, the nucleotide sequence encoding polypeptide chain III is shown as SEQ ID NO:111, and the nucleotide sequence encoding polypeptide chain IV is shown as SEQ ID NO:

112.

50. A vector comprising the nucleic acid molecule of any one of claims 46-49.

51. A cell comprising the antigen binding protein of any one of claims 1-45, the nucleic acid molecule of any one of claims 46-49, and / or the vector of claim 50.

52. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-45 and a pharmaceutically acceptable carrier.

53. Use of the antigen binding protein of any one of claims 1-45 in the preparation of a medicament for treating a tumor or cancer that is positive for MUC1 expression.

54. The use according to claim 53, wherein the MUCl expression-positive tumor or cancer is selected from pancreatic cancer, ovarian cancer, breast cancer, lung adenocarcinoma, fallopian tube cancer and colorectal cancer.

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