Antigen-binding protein targeting MUC1

Abstract

Provided is an antigen-binding protein targeting MUC1. The antigen-binding protein contains at least one CDR in a heavy chain variable region of an antibody. Further provided are a chimeric antigen receptor, immunoconjugate and pharmaceutical composition containing the antigen-binding protein, a nucleic acid encoding the antigen-binding protein, a vector containing the nucleic acid molecule, a cell containing the vector, and the use of the antigen-binding protein in the prevention and / or treatment of diseases.

Description

Antigen binding proteins targeting MUC1 TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an antigen binding protein targeting MUC1 and application thereof. BACKGROUND

[0002] Mucin (MUC1) is a type I transmembrane glycoprotein with a core protein mass of 120-225 kDa, which is increased to 250-500 kDa by glycosylation. MUC1 protein extends 200-500 nanometers beyond the cell surface, anchored on the apical surface of many epithelial cells by a transmembrane domain. MUC1 contains two parts of MUC1-N (alpha subunit) and MUC1-C (beta subunit), the N-terminal contains multiple O-glycosylated repeat sequences (VNTR), and the C-terminal contains an ECD region, a 28 aa transmembrane domain (TMD) and a 72 aa intracellular domain (CT). MUC1 is widely distributed and abnormally abundant on the surface of cancer cells, and abnormally glycosylated MUC1 is commonly found in breast cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, gastric cancer, myeloma, liver cancer and / or bile duct cancer.

[0003] With the occurrence and development of tumors, MUC1 on the surface of tumor cells can be cleaved by a series of proteases such as ADAM, MMP enzymes, etc. in the tumor microenvironment, shedding the alpha subunit and exposing the MUC1 beta subunit. This region can act as a growth factor receptor and bind to NME series ligands to regulate the growth of cancer cells. In addition, the shed alpha subunit, CA153, can be free in the blood as a marker for various cancers such as breast cancer, lung cancer, colon cancer, ovarian cancer, etc. Compared with the cleavage of healthy progenitor cell MUC1, the involved cleavage enzyme and cleavage site can be different, and in addition, the MUC1 beta subunit has an N-glycosylation modification, and abnormal glycosylation can expose new epitopes.

[0004] Therefore, as a target with great development potential, it is urgent to develop more antigen binding proteins that can effectively bind to MUC1 to achieve more effective therapeutic effects on MUC1-related tumors. SUMMARY

[0005] The present application provides an antigen binding protein targeting MUC1 beta subunit. In the present application, the antigen binding protein has one or more of the following properties: (1) capable of specifically binding to MUC1 beta subunit and having good binding activity; (2) capable of specifically binding to MUC1 beta subunit expressed on the surface of target cells; (3) having a killing effect on tumors; (4) having a certain endocytosis effect and having potential to be used as an antibody targeting part of ADC drugs.

[0006] An isolated antigen binding protein capable of binding to MUC1 β subunit, wherein the antigen binding protein comprises an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 186, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187 or SEQ ID NO: 188, the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189 or SEQ ID NO: 190.

[0007] In certain embodiments, the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, the LCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 77-80, SEQ ID NO: 83 or SEQ ID NO: 191, the LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 192, the LCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 90-92, SEQ ID NO: 94 or SEQ ID NO: 193.

[0008] In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3, and the LCDR1, LCDR2 and LCDR3 are selected from any one of the following combinations:

[0009] (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 15, the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 24, the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 77, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 84, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 90;

[0010] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 91;

[0011] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 92;

[0012] (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 93;

[0013] (5) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94;

[0014] (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94;

[0015] (7) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 89, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95; and

[0016] (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0017] In certain embodiments, the amino acid sequences of the VH and VL are selected from any of the following combinations:

[0018] (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 147, the VL comprises the amino acid sequence set forth in SEQ ID NO: 163;

[0019] (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 148, the VL comprises the amino acid sequence set forth in SEQ ID NO: 164;

[0020] (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 149, the VL comprises the amino acid sequence set forth in SEQ ID NO: 165;

[0021] (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 150, the VL comprises the amino acid sequence set forth in SEQ ID NO: 166;

[0022] (5) the VH comprises an amino acid sequence set forth in SEQ ID NO: 151, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 167;

[0023] (6) the VH comprises an amino acid sequence set forth in SEQ ID NO: 152, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 168;

[0024] (7) the VH comprises an amino acid sequence set forth in SEQ ID NO: 153, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 169;

[0025] (8) the VH comprises an amino acid sequence set forth in SEQ ID NO: 154, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 170;

[0026] (9) the VH comprises an amino acid sequence set forth in SEQ ID NO: 155, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 171;

[0027] (10) the VH comprises an amino acid sequence set forth in SEQ ID NO: 156, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 172;

[0028] (11) the VH comprises an amino acid sequence set forth in SEQ ID NO: 157, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 173;

[0029] (12) the VH comprises an amino acid sequence set forth in SEQ ID NO: 158, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 174; and

[0030] (13) the VH comprises an amino acid sequence set forth in SEQ ID NO: 159, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 175.

[0031] In certain embodiments, the antigen binding protein is a scFv comprising an amino acid sequence set forth in any one of SEQ ID NOs: 142-146 or SEQ ID NOs: 194-201.

[0032] In another aspect, the present application provides an isolated antigen binding protein capable of binding to MUC1 β subunit, wherein the antigen binding protein comprises a VH comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 13, and the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22.

[0033] In certain embodiments, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are selected from any one of the following combinations:

[0034] (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 6, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 14, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 23; and

[0035] (2) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 5, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 13, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 22.

[0036] In certain embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO: 160.

[0037] In certain embodiments, the antigen binding protein is a VHH comprising an amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO: 160.

[0038] In another aspect, the present application provides an antigen binding protein targeting MUC1 α subunit. The antigen binding protein described in the present application is humanized from the FR region of the murine 1B2 antibody (SEQ ID NO: 202, expired patent US8722856B2) targeting MUC1 α subunit, without changing the CDR (Kabat coding) sequence, greatly improving its binding affinity to MUC1 α subunit. In the present application, the antigen binding protein has one or more of the following properties: (1) capable of specifically binding to MUC1 α subunit and having good binding activity; (2) capable of specifically binding to MUC1 α subunit expressed on the surface of target cells; (3) having a killing effect on tumors.

[0039] The present application provides an isolated antigen binding protein that is capable of binding to a MUC1 alpha subunit, wherein the antigen binding protein comprises an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising an amino acid sequence set forth in SEQ ID NO: 203.

[0040] In certain embodiments, the VL comprises an amino acid sequence set forth in SEQ ID NO: 210.

[0041] In certain embodiments, the antigen binding protein comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 203 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 210.

[0042] In certain embodiments, the antigen binding protein is a scFv comprising an amino acid sequence set forth in SEQ ID NO: 213.

[0043] In certain embodiments, the antigen binding protein further comprises an immunoglobulin constant region.

[0044] In certain embodiments, the antigen binding protein is an antibody or an antigen binding fragment thereof.

[0045] In certain embodiments, the antigen binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

[0046] In another aspect, the present application provides a chimeric antigen receptor comprising the antigen binding protein.

[0047] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0048] In certain embodiments, the antigen binding domain comprises the antigen binding protein.

[0049] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0050] In certain embodiments, the chimeric antigen receptor comprises a CD8 alpha signal peptide, an antigen binding protein targeting a MUC1 alpha subunit or beta subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0051] In another aspect, the present application provides a modified immune cell comprising the chimeric antigen receptor.

[0052] In certain embodiments, the immune cell is a T cell, an NK cell, an iNKT cell, a dendritic cell, and / or a macrophage.

[0053] In another aspect, the present application provides an immunoconjugate, wherein the immunoconjugate comprises the antigen binding protein.

[0054] In another aspect, the present application provides an isolated nucleic acid molecule encoding the antigen binding protein and / or the chimeric antigen receptor.

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

[0056] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0057] In another aspect, the present application provides a pharmaceutical composition comprising the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, and / or cell, and optionally a pharmaceutically acceptable carrier.

[0058] In another aspect, the present application provides a detection kit comprising the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition, for detecting the presence and / or amount of MUC1 β subunit or MUC1 α subunit in a sample or subject.

[0059] In another aspect, the present application provides a method of detecting the presence and / or amount of MUC1 β subunit or MUC1 α subunit, comprising using the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition.

[0060] In another aspect, the present application provides use of the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a disease and / or disorder.

[0061] In another aspect, the present application provides a method of preventing and / or treating a disease and / or disorder, comprising administering to a subject in need thereof the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition.

[0062] In another aspect, the present application provides an antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition for use in preventing and / or treating a disease and / or disorder.

[0063] In certain embodiments, the disease and / or disorder is a tumor.

[0064] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0065] In certain embodiments, the tumor is positive for MUC1 β subunit or MUC1 α subunit expression.

[0066] In certain embodiments, the tumor is a MUC1 β subunit or MUC1 α subunit positive tumor of epithelial origin.

[0067] In certain embodiments, the tumor is a breast cancer, a prostate cancer, a colorectal cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, a gastric cancer, a myeloma, a liver cancer, and / or a cholangiocarcinoma.

[0068] Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, wherein only the preferred embodiment of the application is shown and described. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the inventive aspects of the application. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0069] The specific features of the application involved are shown in the appended claims. The features and advantages of the application involved can be better understood from the detailed description of the exemplary embodiments and from the accompanying drawings, which are described briefly below:

[0070] Figure 1 shows a graph of the results of a flow cytometry assay of the binding activity of the antibody 1A1 described herein to the MUC1 β subunit.

[0071] Figure 2 shows a graph of the results of a flow cytometry assay of the binding activity of the antibody 1A2 described herein to the MUC1 β subunit.

[0072] Figure 3 shows a graph of the results of a flow cytometry assay of the binding activity of the camelid scFv format antibodies described herein to the MUC1 β subunit.

[0073] Figure 4 shows a graph of the results of a flow cytometry assay of the binding activity of the camelid scFv format antibodies described herein to embryonic stem cells H1.

[0074] Figure 5 shows a graph of the results of a flow cytometry assay of the binding activity of the murine candidate chimeric antibodies described herein to the MUC1 β subunit.

[0075] Figure 6 shows the results of flow cytometry assay of the binding activity of the murine candidate chimeric antibodies described herein to embryonic stem cells Hl.

[0076] Figure 7 shows the results of flow cytometry assay of the internalization of the antibodies described herein.

[0077] Figure 8 shows the structure of the CAR targeting the MUC1 β subunit described herein.

[0078] Figure 9 shows the results of expansion of the CAR-T cells described herein.

[0079] Figure 10 shows the results of the positive rate of CAR expression described herein.

[0080] Figures 11A and 11B show the results of the killing effect of the CAR-T cells described herein on the MUC1 β subunit overexpressing HCT116 tumor cell line; Figures 11C and 11D show the results of the induction of IFN-γ secretion by the CAR-T cells described herein during the killing of target cells.

[0081] Figure 12 shows the results of competitive binding of the antibodies targeting the MUC1 β subunit described herein.

[0082] Figure 13 shows the results of the binding activity of the humanized antibodies targeting the MUC1 β subunit described herein to tumor cell surface MUC1 β subunit.

[0083] Figure 14A shows the results of flow cytometry of the antibody B21 described herein on HCT116 tumor cells; Figure 14B shows the results of flow cytometry of the antibody B21 described herein on CAOV3 ovarian cancer; Figure 14C shows the results of flow cytometry of the antibody B21 described herein on T47D colon cancer.

[0084] Figure 15 shows the results of the specific binding activity of the antibody B21 described herein to MUC1.

[0085] Figure 16 shows the structure of the B21-CAR described herein.

[0086] Figure 17 shows the results of expansion of the B21-CAR-T cells described herein.

[0087] Figure 18 shows the results of the positive rate of B21-CAR expression described herein.

[0088] Figure 19A shows the CD25 expression results of B21-CAR-T and control CAR-T in the presence of different concentrations of CA153; Figure 19B shows the CD69 expression results of B21-CAR-T and control CAR-T in the presence of different concentrations of CA153; Figure 19C shows the CD137 expression results of B21-CAR-T and control CAR-T in the presence of different concentrations of CA153.

[0089] Figure 20A shows the staining intensity results of the target abundance of the advanced patient; Figure 20B shows the positive rate results of the target abundance of the advanced patient.

[0090] Figure 21 shows the CAR structure targeting MUC1 a subunit, targeting MUC1 β subunit, and simultaneously targeting MUC1 a subunit and β subunit.

[0091] Figure 22A-B shows the target cell target abundance results of the present application.

[0092] Figure 23 shows the schematic diagram of the downstream promoter NFAT activating GFP after the CAR-J cells of the present application are stimulated by target cells of different MUC1 forms.

[0093] Figure 24 shows the CAR activation degree results of the present application.

[0094] Figure 25 shows the induced cytokine secretion results of CAR-T cells of the present application killing wild-type SKOV3 in vitro.

[0095] Figure 26 shows the induced cytokine secretion results of CAR-T cells of the present application killing overexpressed cell lines in vitro.

[0096] Figure 27 shows the percentage of target cell killing by CAR-T cells of the present application.

[0097] Figure 28 shows the induced cytokine secretion results of CAR-T cells of the present application killing target cells in vitro.

[0098] Figure 29 shows the positive expression rate results of the CAR of the present application under different CA153 protein concentrations and the activation marker expression results of the CAR-T of the present application under different CA153 protein concentrations.

[0099] Figure 30 shows the positive expression rate results of the CAR of the present application under different sources of CA153 protein and the activation marker expression results of the CAR-T of the present application under different sources of CA153 protein.

[0100] Figure 31 shows the results of tumor growth changes in mice inoculated with CAR-T described in the application. DETAILED DESCRIPTION

[0101] The following examples illustrate the embodiments of the application described in the specification. Other advantages and effects of the application will be readily appreciated by those skilled in the art from the disclosure in the specification.

[0102] Definitions of terms

[0103] In the present application, the terms "mucin 1", "MUC1", "mucin1" and "CD227" can be used interchangeably, and MUC1 generally refers to a member of the mucin family, which is a type I transmembrane glycoprotein. MUC1 has a core protein mass of 120-225 kDa, which is increased to 250-500 kDa by glycosylation. MUC1 protein extends 200-500 nanometers beyond the cell surface, anchored on the apical surface of many epithelial cells by a transmembrane domain. MUC1 contains two parts, MUC1-N (alpha subunit) and MUC1-C (beta subunit), the N-terminal contains multiple O-glycosylated repeat sequences (VNTR), and the C-terminal contains the ECD region, a 28 aa transmembrane domain (TMD) and a 72 aa intracellular domain (CT). The cleaved MUC1 alpha subunit (CA153) can be used as a tumor marker. MUC1 is widely distributed and abnormally abundant on the surface of cancer cells. MUC1 on tumor cells has abnormal O-glycosylation, exposing new peptide epitopes and new carbohydrate tumor antigens on the MUC1 protein backbone, including GalNAcα-O-Ser / threonine (Tn, CD175), Neu5Acα2, 6-GalNAcα-O-Ser / threonine (sTn, CD175s) and other tumor-specific glycosyl antigens. In the present application, the MUC1 can be tumor-associated MUC1 ("TA-MUC1"). For example, the MUC1 can be MUC1 present on tumor cells. For example, the tumor can be breast cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, gastric cancer, myeloma, liver cancer and / or bile duct cancer. In the present application, the MUC1 can be intact MUC1 and functionally active fragments, homologues, analogues, variants or derivatives thereof. For example, the MUC1 can be full-length MUC1, or a truncated MUC1 that retains its functional activity. In the present application, the MUC1 can be of any species origin. For example, the MUC1 can be human MUC1. In the present application, the MUC1 can be wild type or artificially modified. For example, the MUC1 can be a modified MUC1.

[0104] In the present application, the term "antigen binding protein" generally refers to a protein having the ability to bind an antigen. In the present application, the antigen binding protein can comprise an antigen binding moiety and, optionally, a scaffold or framework moiety allowing the antigen binding moiety to adopt a conformation that facilitates the binding of the antigen binding moiety to an antigen. In the present application, the antigen binding protein can be wild-type or artificially engineered. In the present application, the antigen binding protein can comprise a protein scaffold of antibody origin or an alternative protein scaffold or artificial scaffold having grafted CDRs or CDR derivatives, for example. In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof, as well as variants, homologues, derivatives or analogues thereof.

[0105] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof that can have a specific binding reaction with a corresponding antigen. In the present application, the antibody can include a protein of at least two heavy chains and two light chains that are connected to each other by disulfide bonds, or an antigen-binding fragment thereof. In the present application, the antibody can also be a single domain antibody. For example, the single domain antibody can include a heavy chain variable segment VHH. In the present application, the antibody can also be a single chain antibody (scFv). For example, the single chain antibody can include heavy and light chain variable segments. In the present application, the heavy chain can include a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region includes three domains CH1, CH2, and CH3. In the present application, the light chain can include a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region includes one domain. In the present application, the VH and VL regions can include regions of hypervariability, referred to as complementarity determining regions (CDRs) or hypervariable regions (HVRs), which alternate with regions that are more conserved, referred to as framework regions (FRs). In the present application, the VH and VL each include three CDRs and four FRs, arranged in the following order from the amino-terminal to the carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. For example, the variable domains of the heavy and light chains each include four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4). In the present application, the CDRs can be determined by various encoding systems. For example, the CDRs can be determined by CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. For example, the CDRs can be determined by the Kabat encoding system. For example, the CDRs can be determined by the IMGT encoding system. In the present application, the CDRs encompass CDR sequences determined according to any CDR partitioning. In the present application, the CDRs can encompass variants thereof. For example, the amino acid sequence of the CDRs can be substituted, deleted, and / or added with one or more amino acids, for example, 1-30, 1-20, or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the CDRs encompass homologues. For example, the homologues can be amino acid sequences having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology with the amino acid sequence of the CDRs. In the present application, the antibody can be of animal origin or non-animal origin. For example, the antibody can be of murine origin. For example, the antibody can be of camel origin. For example, the antibody can be of human origin.In the present application, the antibody can be artificially synthesized. In the present application, the antibody can be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, or a multispecific antibody. In the present application, the antibody can be a chimeric antibody, a humanized antibody, or a fully human antibody. In the present application, the antibody can be a recombinant, hybrid, mutated, or grafted antibody.

[0106] In the present application, the term "antigen-binding fragment" generally refers to a portion of an intact antibody, and includes the variable region of an intact antibody. For example, the antigen-binding fragment can include a Fab, a (Fab)2, a F(ab')2, a scFv, a di-scFv, a Fv, a VHH, or a dAb fragment. In the present application, the antigen-binding fragment is obtained using conventional techniques known to one of ordinary skill in the art, and the function of the fragment is assessed in the same manner as the intact antibody. For example, the antigen-binding fragment can be obtained by an in vitro display technique.

[0107] In the present application, the terms "VHH", "nanobody", and "single-domain antibody" can be generally used interchangeably, and generally refer to an antibody structure consisting of a heavy chain variable region. In the present application, the VHH can include a complementarity determining region (CDR) and a framework region (FR). In the present application, the VHH can include three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0108] In the present application, the terms "scFv" and "single-chain antibody" can be generally used interchangeably, and generally refer to an antibody structure consisting of a heavy chain variable region and a light chain variable region. In the present application, the heavy chain variable region and the light chain variable region can include a complementarity determining region (CDR) and a framework region (FR). In the present application, the heavy chain variable region and the light chain variable region can include three CDRs and four FRs, respectively, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In the present application, the heavy chain variable region and the light chain variable region of the scFv can be connected by a linker. For example, the linker can be a flexible linker.

[0109] In the present application, the terms "immunoglobulin constant region" and "constant region" can be used interchangeably, and generally refer to the region of an antibody that is not variable. In the present application, the constant region is not directly involved in binding to an antigen, but can exhibit various effector functions. In the present application, the constant region can be the entire non-variable region of an antibody, or can be a portion of the non-variable region of an antibody. For example, the constant region can be a heavy chain constant region. For example, the constant region can be a light chain constant region. For example, the constant region can be an Fc region. In the present application, the antibody heavy chain constant region can be an IgG heavy chain constant region. For example, the IgG heavy chain constant region can be a heavy chain constant region of IgG1, IgG2, IgG3, or IgG4. In the present application, the antibody light chain constant region can be a kappa (K) or lambda (L) light chain constant region. In the present application, the constant region can be derived from an animal. For example, the constant region can be derived from a human, a goat, a rabbit, a rat, or a guinea pig. For example, the heavy chain constant region can be a human IgG heavy chain constant region. For example, the human IgG heavy chain constant region can be a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the light chain constant region can be a human Kappa or Lambda light chain constant region. For example, the Fc region can be a human Fc region.

[0110] In the present application, the protein and / or amino acid sequence referred to herein should also be understood to encompass variants or homologues of the protein having the same or similar function. In the present application, the variant can be a protein or polypeptide having one or more amino acid substitutions, deletions or additions in the amino acid sequence of the protein (e.g., an antigen binding protein as described herein). For example, the functional variant can comprise a protein or polypeptide having amino acid changes by at least 1, for example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially maintain the biological properties of the protein or polypeptide before the changes (e.g., substitutions, deletions or additions). For example, the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the changes. For example, the substitution can be a conservative substitution.

[0111] In the present application, a portion of the amino acid sequence of the antigen binding protein can be homologous to the corresponding amino acid sequence in an antibody from a particular species, or belong to a particular class. For example, both the variable region and the constant portion of the antibody can be derived from the variable region and the constant region of an antibody from an animal species (e.g., a human).

[0112] In the present application, the homologues can be proteins or polypeptides having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen binding protein described in the present application).

[0113] In the present application, the terms “chimeric antigen receptor” and “CAR” can be used interchangeably, which generally refers to a receptor artificially modified to be able to anchor specific molecules recognizing target cell surface antigens on immune cells, so that the immune cells can recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. For example, the specific molecules can be antigen binding proteins. For example, the immune cells can be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, gd T cells or DNT cells. In the present application, the CAR can comprise, in order, an optional signal peptide, an antigen binding domain, a hinge region, a transmembrane domain region and an intracellular signaling domain. For example, the CAR can further comprise an optional costimulatory domain. In the present application, the antigen binding domain can be secreted by immune cells or artificially synthesized. In the present application, the antigen binding domain can be an antigen binding protein. In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof. For example, the antigen binding fragment can be a Fab, a (Fab)2, a F(ab’)2, a scFv, a di-scFv, a Fv, a VHH or a dAb fragment.

[0114] In the present application, the “binding element” generally refers to an element capable of specifically recognizing and binding to an antigen or a target. For example, the binding element can be a polypeptide. For example, the polypeptide can be a natural polypeptide or an artificially synthesized polypeptide. For example, the binding element can be a fusion protein. For example, the binding element can be an antibody or an antigen binding fragment thereof. For example, the antigen binding fragment can be a Fab, a F(ab’)2, a Fv, a scFv or a VHH. In the present application, the binding element can specifically bind to a target antigen. For example, the target antigen can be a tumor antigen. For example, the target antigen can be a tumor associated antigen (TAA). For example, the target antigen can be MUC1. For example, the target antigen can be MUC1 a subunit. For example, the target antigen can be MUC1 b subunit. In the present application, the target antigen can be a shed tumor antigen. For example, the target antigen can be a shed MUC1 a subunit (CA153).

[0115] In the present application, the "transmembrane domain" generally refers to a domain of a peptide, polypeptide, or protein that is capable of spanning the plasma membrane of a cell, which can be used to anchor an extracellular domain to the cell membrane. For example, the transmembrane domain can comprise a transmembrane domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain can be derived from the transmembrane domain of CD8.

[0116] In the present application, the "hinge region" generally refers to a flexible polypeptide. In the present application, the hinge region can allow independent movement of the antigen binding region. For example, the hinge region can comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region can be derived from the hinge region of CD8.

[0117] In the present application, the "intracellular signaling domain" generally refers to an intracellular region that can generate a signal that promotes the immune effector function of a CAR-containing cell. For example, the intracellular signaling domain can comprise an intracellular signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon RI gamma, Fc epsilon RI beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling domain can be a signaling domain derived from CD3 zeta.

[0118] In the present application, the "co-stimulatory domain" generally refers to a part of the CAR that is capable of transducing an effector signal. For example, the co-stimulatory domain can include a co-stimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon R1 gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40, and MyD88. For example, the co-stimulatory domain can be a co-stimulatory domain derived from 4-1BB.

[0119] In the present application, the term "shedding" generally refers to a process in which a polypeptide is released into blood, body fluid, and tissue after being produced by a cell. For example, the shedding can refer to a process in which a polypeptide is released into blood, body fluid, and tissue after being hydrolytically cleaved by a protease. In the present application, the polypeptide can be an antigen. For example, the polypeptide can be a cell surface antigen. For example, the polypeptide can be a tumor antigen. For example, the tumor antigen can be MUC1. In the present application, the shedding can be a process in which a tumor marker (cancer marker) is released after being synthesized by a tumor cell during tumor occurrence and proliferation. For example, the tumor marker can be CA153.

[0120] In the present application, the terms "tandem repeat sequence" and "VNTR" can be used interchangeably, and the tandem repeat sequence generally refers to a nucleotide sequence in DNA or an amino acid sequence in a protein having a plurality of repeat units connected in front and behind. For example, the tandem repeat sequence can be an amino acid sequence in a protein having a plurality of repeat units connected in front and behind. In the present application, the tandem repeat sequence has three or more repeat units connected in front and behind. In the present application, the tandem repeat sequence can be a tandem repeat sequence of an antigen. For example, the tandem repeat sequence can be a tandem repeat sequence of a tumor antigen. For example, the tandem repeat sequence can be a tandem repeat sequence of the MUC1 alpha subunit. In the present application, the tandem repeat sequence can be a full-length tandem repeat sequence or a truncated tandem repeat sequence in a protein. In the present application, the tandem repeat sequence can be divided in any manner as long as the repeat units are connected in front and behind.

[0121] In the present application, the term "multimerization" generally refers to a process that, after a ligand binds to a receptor, multiple receptor molecules are brought close to each other to interact. For example, the multimerization can refer to a process that, after a CAR specifically binds to an antigen, multiple CARs are brought close to each other to interact. For example, the multimerization can refer to a process that, after a CAR specifically binds to an antigen, intracellular signaling domains of multiple CARs are brought close to each other to activate and / or stimulate signaling. In the present application, the antigen can be a tumor antigen. For example, the multimerization can refer to a process that, after a CAR specifically binds to a tumor antigen, intracellular signaling domains of multiple CARs are brought close to each other to activate and / or stimulate signaling. For example, the multimerization can refer to a process that, after a CAR specifically binds to a tumor antigen having tandem repeats, intracellular signaling domains of multiple CARs are brought close to each other to activate and / or stimulate signaling. In the present application, the multimerization can result from multiple CARs simultaneously binding to the same antigen. For example, the multimerization can result from multiple CARs simultaneously binding to the same tumor antigen. For example, the multimerization can result from multiple CARs simultaneously binding to the tandem repeats of the same tumor antigen. For example, the multimerization can result from multiple CARs simultaneously binding to the tandem repeats of CA153.

[0122] In the present application, the terms "expansion" and "proliferation" can be used interchangeably, and expansion generally refers to a process that a starting cell generates multiple cell individuals in a divisional manner. In the present application, the multiple cell individuals can be the same type of cells or different types of cells. In the present application, the starting cell for expansion does not need to be the same as the cell generated by expansion. For example, the expanded cells can be generated from the growth and differentiation of a starting population of cells.

[0123] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating an active ingredient to the antigen-binding protein. In the present application, the active ingredient can be covalently linked to the antigen-binding protein through a linker molecule. In the present application, the immunoconjugate can be an antigen-binding protein conjugated with a payload. For example, the payload can be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid. In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a radiopharmaceutical (RDC). In the present application, the conjugate can deliver the payload to a target cell through specific binding of the antigen-binding protein to an antigen on the target cell. For example, the target cell can be a tumor cell.

[0124] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a nontoxic material that does not interfere with the effectiveness or the biological activity of the active ingredients. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is nontoxic to the cells or mammals exposed thereto at the employed doses and concentrations. For example, the physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid, or inactivated viral particle.

[0125] In the present application, the term "preventing and / or treating" generally refers to preventing and / or treating a disease. For example, the preventing and / or treating can be preventing the onset of the disease, slowing down or reversing the disease progression, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In the present application, the disease can be a tumor disease. For example, preventing or alleviating the onset of one or more symptoms associated with a tumor, reducing the severity and duration of a tumor and symptoms associated therewith.

[0126] In the present application, the term "tumor" generally refers to any new pathological tissue proliferation, which presents a tumor antigen that can be recognized by the immune system. In the present application, the tumor can include a benign or malignant tumor (cancer). In the present application, the cancer can be a metastatic cancer and a non-metastatic cancer. In the present application, the tumor can include a solid tumor and / or a hematological tumor. In the present application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor can include a neoplasm or a solid lesion formed by abnormal cell growth. In the present application, the hematological tumor generally refers to a class of diseases of the hematopoietic system. In the present application, the hematological tumor can include various types of leukemia, multiple myeloma, or malignant lymphoma. In the present application, the tumor can be a tumor positive for MUC1 β subunit expression. In the present application, the tumor can be a tumor positive for MUC1 α subunit expression. For example, the tumor can be breast cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, gastric cancer, myeloma, liver cancer, and / or bile duct cancer.

[0127] In the present application, the term "subject" generally refers to a human or a non-human animal, including but not limited to a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, or a monkey.

[0128] In the present application, the term "comprising" generally refers to including the explicitly specified features, but not excluding other elements.

[0129] In the present application, the term "about" generally refers to a variation within a range of 0.5-10% above or below the specified numerical value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0130] DETAILED DESCRIPTION

[0131] Antigen binding protein

[0132] The CDRs of an antibody, also known as complementarity determining regions, are portions of the variable region. The amino acid residues of this region can contact the antigen or epitope. Antibody CDRs can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. These numbering systems are known in the art, and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. One of skill in the art can determine the CDR regions from the sequence and structure of an antibody using different numbering systems. The CDR regions can differ using different numbering systems. In the present application, the CDRs encompass CDR sequences determined according to any CDR partitioning scheme; also encompass variants thereof, which include substitution, deletion, and / or addition of one or more amino acids of the CDR. For example, 1-30, 1-20, or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; also encompass homologues thereof, which can be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CDR. For example, the CDRs of an antigen binding protein in the present application can be determined using IMGT. For example, the CDRs of an antigen binding protein in the present application can be determined using Kabat.

[0133] In one aspect, the present application provides an isolated antigen binding protein capable of specifically binding to MUC1 β subunit, wherein the antigen binding protein comprises at least one CDR in an antibody heavy chain variable region. In the present application, the antigen binding protein targeting MUC1 β subunit can comprise an antibody heavy chain variable region VH, which comprises heavy chain complementarity determining regions HCDR1, HCDR2, and / or HCDR3.

[0134] In the present application, the antigen binding protein targeting the MUC1 β subunit has one or more of the following properties: (1) capable of specifically binding to the MUC1 β subunit and has good binding activity; (2) capable of specifically binding to the MUC1 β subunit expressed on the surface of target cells; (3) has a killing effect on tumors; (4) has a certain endocytosis effect and can be potentially used as an antibody targeting moiety of ADC drugs. For example, the antigen binding protein can bind to the MUC1 β subunit on the surface of target cells with higher affinity / binding activity than the control antibody H44. For example, the antigen binding protein can bind to the MUC1 β subunit with a lower KD than the control antibody. For example, the antigen binding protein can have endocytosis superior to or equivalent to the control antibody H44.

[0135] In the present application, the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190. In the present application, the HCDR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

[0136] In the present application, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188. In the present application, the HCDR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15-21.

[0137] In the present application, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186. In the present application, the HCDR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 7-12.

[0138] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, and the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190.

[0139] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, and the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190.

[0140] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, the HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-21, and the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190.

[0141] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, the HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

[0142] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, the HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-21, the HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

[0143] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, the HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

[0144] In the present application, the VH can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-21, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190.

[0145] In the present application, the VH can comprise heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, and HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

[0146] For example, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 described in the present application can be selected from any one of the following combinations:

[0147] (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 15, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 24;

[0148] (2) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 16, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 25;

[0149] (3) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 17, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 26;

[0150] (4) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 27;

[0151] (5) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 19, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 28;

[0152] (6) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 19, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 29;

[0153] (7) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 20, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 30; and

[0154] (8) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 31.

[0155] In the present application, X1YGMS (SEQ ID NO: 186), wherein X1may be N, R or S. In the present application, VIASDGRTYX1ADSMRG (SEQ ID NO: 187), wherein X1may be F or Y. In the present application, X1ISX2GGX3YTYYPDSVKG (SEQ ID NO: 188), wherein X1may be S or T; X2may be G or N; X3may be N, R or S. In the present application, GFX1MX2Y (SEQ ID NO: 189), wherein X1may be F or Y; X2may be D or E. In the present application, HEFX1X2TVAFDY (SEQ ID NO: 190), wherein X1may be I or T; X2may be I or T

[0156] In the present application, the VH can comprise a H-FR1, a H-FR2, a H-FR3 and / or a H-FR4. In the present application, the VH can comprise a H-FR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 34-44. In the present application, the VH can comprise a H-FR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 47-55. In the present application, the VH can comprise a H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 58-70. In the present application, the VH can comprise a H-FR4 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 72-76.

[0157] In the present application, the H-FR1, the H-FR2, the H-FR3 and the H-FR4 can be selected from any one of the following combinations:

[0158] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 58, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 73;

[0159] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 48, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 59, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 72;

[0160] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 35, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 48, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 60, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74;

[0161] (4) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 61, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 73;

[0162] (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 62, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74;

[0163] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 38, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 50, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 63, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 75;

[0164] (7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 39, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 51, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 64, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74;

[0165] (8) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 52, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 65, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 76;

[0166] (9) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 52, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 66, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 76;

[0167] (10) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 51, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74;

[0168] (11) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 68, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 75;

[0169] (12) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 43, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 69, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74; and

[0170] (13) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:55, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:70, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:75.

[0171] In the present application, the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs:147-159. For example, the VH may be a wild-type sequence. For example, one or more amino acid sequences in the VH may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to the MUC1β subunit. For example, the VH may be a variant thereof, comprising a substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the FR. For example, the VH may also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in any one of SEQ ID NOs:147-159.

[0172] In the present application, the antigen-binding protein may further comprise at least one CDR in an antibody light chain variable region. In the present application, the antigen-binding protein may comprise an antibody light chain variable region VL, wherein the VL comprises light chain complementary determining regions LCDR1, LCDR2 and / or LCDR3.

[0173] In the present application, the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 90-92, SEQ ID NO: 94 or SEQ ID NO: 193. In the present application, the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 90-96.

[0174] In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO: 192. In the present application, the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 84-89.

[0175] In the present application, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 77-80, SEQ ID NO: 83 or SEQ ID NO: 191. In the present application, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 77-83.

[0176] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-80, SEQ ID NO:83 or SEQ ID NO:191, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:192, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-92, SEQ ID NO:94 or SEQ ID NO:193.

[0177] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-83, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:192, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-92, SEQ ID NO:94 or SEQ ID NO:193.

[0178] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-80, SEQ ID NO:83 or SEQ ID NO:191, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:84-89, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-92, SEQ ID NO:94 or SEQ ID NO:193.

[0179] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-80, SEQ ID NO:83 or SEQ ID NO:191, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:192, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-96.

[0180] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-80, SEQ ID NO:83 or SEQ ID NO:191, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:84-89, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-96.

[0181] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-83, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:192, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-96.

[0182] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-83, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:84-89, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-92, SEQ ID NO:94 or SEQ ID NO:193.

[0183] In the present application, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:77-83, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:84-89, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:90-96.

[0184] For example, the amino acid sequences of LCDR1, LCDR2 and LCDR3 described in the present application can be selected from any one of the following combinations:

[0185] (1) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 84, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 90;

[0186] (2) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 91;

[0187] (3) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 92;

[0188] (4) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 93;

[0189] (5) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 94;

[0190] (6) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 82, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 94;

[0191] (7) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 83, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 89, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 95; and

[0192] (8) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 96.

[0193] In the present application, RSSTGAVTTX1NYAN (SEQ ID NO: 191), wherein X1 can be N or S. In the present application, KVSX1RX2S (SEQ ID NO: 192), wherein X1 can be N or Y; X2 can be A or F. In the present application, X1QGX2HX3PX4T (SEQ ID NO: 193), wherein X1 can be F or W; X2 can be S or T; X3 can be F or V; X4 can be L or R.

[0194] In the present application, the VL may comprise L-FR1, L-FR2, L-FR3 and / or L-FR4. In the present application, the VL may comprise L-FR1, and the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:97-108. In the present application, the VL may comprise L-FR2, and the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:109-118. In the present application, the VL may comprise L-FR3, and the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:119-130. In the present application, the VL may comprise L-FR4, and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs:131-139.

[0195] In the present application, the L-FR1, L-FR2, L-FR3 and L-FR4 are selected from any one of the following combinations:

[0196] (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 97, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 109, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 119, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 131;

[0197] (2) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 98, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 110, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 120, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 131;

[0198] (3) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 99, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 111, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 121, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 132;

[0199] (4) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 100, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 112, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 122, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 131;

[0200] (5) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 101, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 112, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 123, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 132;

[0201] (6) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 102, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 113, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 124, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 133;

[0202] (7) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 103, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 114, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 125, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 134;

[0203] (8) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 104, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 115, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 126, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 135;

[0204] (9) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 105, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 115, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 126, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 135;

[0205] (10) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 106, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 116, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 127, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 136;

[0206] (11) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 107, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 117, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 128, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 137;

[0207] (12) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 108, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 118, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 129, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 138; and

[0208] (13) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 102, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 113, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 130, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 139.

[0209] In the present application, the VL can comprise an amino acid sequence set forth in any one of SEQ ID NOs: 163-175. For example, the VL can be a wild-type sequence. For example, one or more amino acid sequences in the VL can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to the MUC1 beta subunit. For example, the VL can be a variant thereof comprising substitution, deletion, and / or addition of one or more amino acids to the amino acid sequences of the FRs. For example, the VL can also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to the amino acid sequence set forth in any one of SEQ ID NOs: 163-175.

[0210] For example, the amino acid sequences of the HCDR1, HCDR2 and HCDR3, and the LCDR1, LCDR2 and LCDR3 described in the present application can be selected from any one of the following combinations:

[0211] (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 84, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 90;

[0212] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 91;

[0213] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 92;

[0214] (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 93;

[0215] (5) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94;

[0216] (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94;

[0217] (7) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 89, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95; and

[0218] (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0219] For example, the amino acid sequences of the VH and VL described herein can be selected from any of the following combinations:

[0220] (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 147 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 163;

[0221] (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 148 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 164;

[0222] (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 149 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 165;

[0223] (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 150 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 166;

[0224] (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 151 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 167;

[0225] (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 152 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 168;

[0226] (7) the VH comprises the amino acid sequence set forth in SEQ ID NO: 153 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 169;

[0227] (8) the VH comprises the amino acid sequence set forth in SEQ ID NO: 154 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 170;

[0228] (9) the VH comprises an amino acid sequence set forth in SEQ ID NO: 155, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 171;

[0229] (10) the VH comprises an amino acid sequence set forth in SEQ ID NO: 156, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 172;

[0230] (11) the VH comprises an amino acid sequence set forth in SEQ ID NO: 157, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 173;

[0231] (12) the VH comprises an amino acid sequence set forth in SEQ ID NO: 158, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 174; and

[0232] (13) the VH comprises an amino acid sequence set forth in SEQ ID NO: 159, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 175.

[0233] For example, the antigen binding protein can comprise antibody heavy and light chains, the amino acid sequences of which can be selected from any of the following combinations:

[0234] (1) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 181, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 176;

[0235] (2) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 182, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 177;

[0236] (3) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 183, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 178;

[0237] (4) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 184, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 179; and

[0238] (5) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 185, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 180.

[0239] In the present application, the antigen binding fragment can be a scFv comprising a VH and a VL, which are directly or indirectly linked. For example, the VH and the VL are linked by a linker. For example, the linker can be (GGGGS)3(SEQ ID NO: 216). For example, the antigen binding fragment can be a scFv, which can comprise an amino acid sequence set forth in any one of SEQ ID NOs: 142-146 or SEQ ID NOs: 194-201.

[0240] In another aspect, the present application provides an isolated antigen binding protein capable of binding to the MUC1 β subunit, wherein the antigen binding protein comprises at least one CDR in an antibody heavy chain variable region.

[0241] In the present application, the antigen binding protein can comprise a VHH comprising a heavy chain complementarity determining region HCDR1, HCDR2 and / or HCDR3.

[0242] In the present application, the HCDR3 can comprise an amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22. In the present application, the HCDR2 can comprise an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 13. In the present application, the HCDR1 can comprise an amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5.

[0243] For example, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 described in the present application can be selected from any one of the following combinations:

[0244] (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 6, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 14, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 23; and

[0245] (2) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 5, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 13, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 22.

[0246] In the present application, the VHH can comprise an H-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 32. In the present application, the VHH can comprise an H-FR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 45. In the present application, the VHH can comprise an H-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 56. In the present application, the VHH can comprise an H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 72 or SEQ ID NO: 71.

[0247] For example, the VHH described in the present application can comprise an H-FR1, an H-FR2, an H-FR3 and an H-FR4 selected from any one of the following combinations:

[0248] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 46, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 57, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 72; and

[0249] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 45, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 56, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 71.

[0250] In the present application, the VHH can comprise the amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO: 160. For example, the VHH can be a wild-type sequence. For example, one or more amino acid sequences in the VHH can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to the MUC1 β subunit. For example, the VHH can be a variant thereof, which includes substitution, deletion and / or addition of one or more amino acids to the amino acid sequence of the FR. For example, the VHH can also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to the amino acid sequence set forth in any one of SEQ ID NO: 161 or SEQ ID NO: 160.

[0251] In another aspect, the present application provides an isolated antigen binding protein capable of specifically binding to MUC1 a subunit, wherein the antigen binding protein comprises an antibody heavy chain variable region VH, which comprises a HCDR1, a HCDR2 and a HCDR3, the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 205, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 206.

[0252] In the present application, the antigen binding protein targeting MUC1 a subunit has one or more of the following properties: (1) capable of specifically binding to MUC1 a subunit, and has good binding activity; (2) capable of specifically binding to MUC1 a subunit expressed on the surface of target cells; (3) has a killing effect on tumors.

[0253] In the present application, the antigen binding protein has the same CDR (Kabat coding) as the 1B2 antibody (lapsed patent US8722856B2), and only the optimization of the FR region greatly improves its binding affinity to MUC1 a subunit.

[0254] In the present application, the VH can comprise H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the VH can comprise H-FR1, which comprises an amino acid sequence set forth in SEQ ID NO: 207. In the present application, the VH can comprise H-FR2, which comprises an amino acid sequence set forth in SEQ ID NO: 208. In the present application, the VH can comprise H-FR3, which comprises an amino acid sequence set forth in SEQ ID NO: 209. In the present application, the VH can comprise H-FR4, which comprises an amino acid sequence set forth in SEQ ID NO: 74.

[0255] In the present application, the VH can comprise H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 207, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 208, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 209, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 74.

[0256] In the present application, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 203. For example, the VH can be a wild-type sequence. For example, one or more amino acid sequences in the VH can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to the MUC1 alpha subunit. For example, the VH can be a variant thereof that includes substitution, deletion, and / or addition of one or more amino acids to the amino acid sequences of the FRs. For example, the VH can also comprise a sequence that has at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 203.

[0257] In the present application, the antigen binding protein can also comprise a light chain variable region VL that comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. In the present application, the antigen binding protein can also comprise a light chain variable region VL that comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 211.

[0258] In the present application, the VL can comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. In the present application, the VL can comprise L-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 103. In the present application, the VL can comprise L-FR2 comprising the amino acid sequence set forth in SEQ ID NO: 114. In the present application, the VL can comprise L-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 125. In the present application, the VL can comprise L-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 212.

[0259] In the present application, the VL can comprise L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 103, the L-FR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, the L-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 125, and the L-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 212.

[0260] In the present application, the antigen binding protein can comprise L-FR1, L-FR2, L-FR3 and L-FR4. In the present application, the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1, the L-FR2 can be located between the LCDR1 and the LCDR2, the L-FR3 can be located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 can be directly or indirectly connected to the C-terminus of the LCDR3.

[0261] In the present application, the isolated antigen binding protein can comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4, and the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1, the L-FR2 can be located between the LCDR1 and the LCDR2, the L-FR3 can be located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 can be directly or indirectly connected to the C-terminus of the LCDR3.

[0262] In the present application, the VL can comprise the amino acid sequence set forth in SEQ ID NO: 210. For example, the VL can be a wild-type sequence. For example, one or more amino acid sequences in the VL can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to the MUC1 alpha subunit. For example, the VL can be a variant thereof, which includes substitution, deletion and / or addition of one or more amino acids to the amino acid sequence of the FR. For example, the VL can also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 210.

[0263] In the present application, the antigen binding protein can comprise VH and VL, the VH can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 205, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 206; the VL can comprise LCDR1, LCDR2 and LCDR3, in the present application, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 211.

[0264] In the present application, the antigen binding protein can comprise a VH comprising an amino acid sequence set forth in SEQ ID NO: 203 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 210.

[0265] In the present application, the antigen binding fragment can be a scFv comprising a VH and a VL linked directly or indirectly. For example, the VH and the VL are linked by a linker. For example, the linker can be (GGGGS)3. For example, the antigen binding fragment can be a scFv comprising an amino acid sequence set forth in SEQ ID NO: 213.

[0266] In the present application, the EC50value or KDvalue can be determined by routine skill in the art. In the present application, the KDvalue can be determined by Octet, SPR, ELISA, competitive ELISA or BIACORE or KINEXA. In the present application, the antigen binding protein is capable of specifically binding to DLL3. In the present application, the specific binding can be determined by FACS.

[0267] In the present application, the antigen binding protein can comprise a H-FR1, a H-FR2, a H-FR3 and a H-FR4. In the present application, the C-terminus of the H-FR1 can be directly or indirectly linked to the N-terminus of the HCDR1, the H-FR2 can be located between the HCDR1 and the HCDR2, the H-FR3 can be located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 can be directly or indirectly linked to the C-terminus of the HCDR3.

[0268] In the present application, the antigen binding protein can comprise a HCDR1, a HCDR2, a HCDR3, a H-FR1, a H-FR2, a H-FR3 and / or a H-FR4, and the C-terminus of the H-FR1 can be directly or indirectly linked to the N-terminus of the HCDR1, the H-FR2 can be located between the HCDR1 and the HCDR2, the H-FR3 can be located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 can be directly or indirectly linked to the C-terminus of the HCDR3.

[0269] In the present application, the antigen binding protein can comprise L-FR1, L-FR2, L-FR3 and L-FR4. In the present application, the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1, the L-FR2 can be located between the LCDR1 and the LCDR2, the L-FR3 can be located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 can be directly or indirectly connected to the C-terminus of the LCDR3.

[0270] In the present application, the antigen binding protein can comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and / or L-FR4, and the C-terminus of the L-FR1 can be directly or indirectly connected to the N-terminus of the LCDR1, the L-FR2 can be located between the LCDR1 and the LCDR2, the L-FR3 can be located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 can be directly or indirectly connected to the C-terminus of the LCDR3.

[0271] In the present application, the antigen binding protein can further comprise an immunoglobulin constant region. In the present application, the immunoglobulin constant region can be a heavy chain constant region of IgG and / or a light chain constant region of an antibody. In the present application, the heavy chain constant region of IgG can be a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region of IgG can be a heavy chain constant region of IgG1. In the present application, the light chain constant region of the human antibody can be a Kappa or Lambda light chain constant region. In the present application, the immunoglobulin constant region can be an immunoglobulin Fc region. For example, the immunoglobulin constant region can be a wild-type sequence or can be mutated or optimized. For example, the immunoglobulin constant region can be an IgG Fc region. For example, the immunoglobulin constant region can be an IgG1 Fc region. For example, the immunoglobulin constant region can be an IgG4 Fc region. In the present application, the immunoglobulin constant region can be a wild-type sequence or can be mutated or optimized.

[0272] In the present application, the immunoglobulin constant region can be an immunoglobulin constant region of any species origin. For example, the immunoglobulin constant region can be an immunoglobulin constant region of murine, rabbit, goat, or human origin. For example, the immunoglobulin constant region can be a human immunoglobulin constant region. In the present application, the immunoglobulin constant region can be a heavy chain constant region of human IgG and / or a light chain constant region of human antibody. In the present application, the heavy chain constant region of human IgG can be a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the heavy chain constant region of human IgG can be a human IgG1 heavy chain constant region. In the present application, the light chain constant region of human antibody can be a human Kappa or Lambda light chain constant region. For example, the Fc region can be derived from a murine, rabbit, goat, llama, or human Fc region. For example, the immunoglobulin constant region can be a human IgG Fc region.

[0273] In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof. For example, the antigen binding protein described in the present application can include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a single chain antibody, a multi-antibody, an Fv fragment, an scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single domain antibody.

[0274] In the present application, the antigen binding protein can be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen binding protein is mutated / optimized for FR and constant region without reducing the binding activity / affinity of the antigen binding protein to the MUC1 β subunit and / or the MUC1 α subunit. For example, the antigen binding protein has lower immunogenicity without reducing the binding activity / affinity of the antigen binding protein to the MUC1 β subunit and / or the MUC1 α subunit.

[0275] In the present application, the antigen binding fragment can be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

[0276] In the present application, the antigen binding protein can be a monospecific antibody, a bispecific antibody, or a multispecific antibody. For example, the antigen binding protein can further comprise a second domain. For example, the antigen binding protein can further comprise a third domain. In the present application, the antigen binding protein can be a monovalent antibody, a bivalent antibody, or a multivalent antibody. For example, the antigen binding protein has one antigen binding site. For example, the antigen binding protein has two antigen binding sites. For example, the antigen binding protein has multiple antigen binding sites.

[0277] In the present application, the direct or indirect connection can be connected by intermolecular forces, or can be connected by a linker.

[0278] In the present application, the amino acid sequence of the FR can be of any species origin. For example, the FR can be of murine, rabbit, goat, llama, or human origin. In the present application, the amino acid sequence of the FR can be adjusted as needed. For example, the FR can be of wild-type sequence. For example, during the process of humanization of the antibody, the amino acid sequence of the FR can be changed without reducing the binding activity / affinity of the antigen binding protein to the MUC1 β subunit and / or the MUC1 α subunit. For example, during the process of humanization of the antibody, one or more amino acid sequences in the FR can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to the MUC1 β subunit and / or the MUC1 α subunit. For example, the FR can be a variant thereof, which includes substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the FR. For example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the FR.

[0279] In the present application, the antigen binding protein can be expressed in the form of a fusion protein. For example, the antigen binding protein can be fused to a peptide, a polypeptide, an amino acid, or a protein. In the present application, the fusion protein can be a Fab fusion protein, an Fc fusion protein, or a single-chain antibody fusion protein. For example, the antigen binding protein can be fused to one or more functional molecules. For example, the functional molecule can be one or more antigen binding proteins. For example, the functional molecule can be one or more Fc regions.

[0280] In the present application, the antigen binding protein can be wild type. For example, the antigen binding protein can be produced by an immune cell, such as a B cell. For example, the antigen binding protein can comprise a wild type sequence. In the present application, the antigen binding protein can be recombinant. For example, the antigen binding protein can be an antibody produced using recombinant DNA technology. In the present application, the antigen binding protein can be modified. For example, the antigen binding protein can be mutated or optimized. For example, the antigen binding protein can have one or more amino acids substituted, deleted and / or added. For example, 1-30, 1-20 or 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions.

[0281] Chimeric antigen receptor

[0282] In another aspect, the present application provides a chimeric antigen receptor (CAR), which can comprise the antigen binding protein.

[0283] In the present application, the chimeric antigen receptor can comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain. In the present application, the chimeric antigen receptor can further comprise a hinge region. In the present application, the chimeric antigen receptor can further comprise a costimulatory domain.

[0284] In the present application, the antigen binding domain can comprise the aforementioned antigen binding protein. In the present application, the antigen binding domain can target the MUC1 β subunit and / or the MUC1 α subunit.

[0285] In the present application, the transmembrane domain can comprise a transmembrane domain derived from CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM. In the present application, the transmembrane domain can be derived from a transmembrane domain of CD8. In the present application, the amino acid sequence of the CD8 transmembrane domain can be as set forth in SEQ ID NO: 4.

[0286] In the present application, the co-stimulatory domain can comprise a co-stimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon R gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40, and MyD88. In the present application, the co-stimulatory domain can be derived from a 4-1BB co-stimulatory domain. In the present application, the amino acid sequence of the 4-1BB co-stimulatory domain can be as set forth in SEQ ID NO: 1.

[0287] In the present application, the intracellular signaling domain can comprise an intracellular signaling domain derived from CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon R gamma, Fc epsilon R beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, or a domain comprising at least one ITAM. In the present application, the intracellular signaling domain can be derived from a CD3 zeta intracellular signaling domain. In the present application, the amino acid sequence of the CD3 zeta intracellular signaling domain can be as set forth in SEQ ID NO: 2.

[0288] In the present application, the hinge region can comprise a hinge region derived from CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon R gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In the present application, the hinge region can be derived from a CD8 hinge region. In the present application, the amino acid sequence of the CD8 hinge region can be as set forth in SEQ ID NO: 3.

[0289] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 β subunit, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 α subunit, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 β subunit and the MUC1 α subunit, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0290] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 β subunit comprising the foregoing antigen binding protein, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising heavy chain complementarity determining regions HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, and HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190, and a light chain variable region VL. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising heavy chain complementarity determining regions HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, and HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-31, and a light chain variable region VL. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising heavy chain complementarity determining regions HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, and HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-31, LCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 77-83, LCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 84-89, and LCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 90-96, and a light chain variable region VL. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 147-159, and a light chain variable region VL.For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 147-159 and a light chain variable region VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 163-175.

[0291] For example, the chimeric antigen receptor can comprise a scFv targeting the MUC1 β subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the scFv can comprise an amino acid sequence set forth in any one of SEQ ID NOs: 142-146 or SEQ ID NOs: 194-201.

[0292] For example, the chimeric antigen receptor can comprise a Fab targeting the MUC1 β subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the Fab can be selected from a chimeric antibody, which can comprise an antibody heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 181-185 and a light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 176-180.

[0293] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 β subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the antigen binding domain can comprise a VHH comprising a heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 13, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22.

[0294] For example, the chimeric antigen receptor can comprise a VHH targeting the MUC1 β subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the VHH can comprise an amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO: 160.

[0295] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting the MUC1 alpha subunit comprising the foregoing antigen binding protein, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising heavy chain complementarity determining regions HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a light chain variable region VL. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising heavy chain complementarity determining regions HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a light chain variable region VL comprising light chain complementarity determining regions LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 80, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 87, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 211. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 203. For example, the antigen binding domain can comprise an antibody heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 203, and a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 210.

[0296] For example, the chimeric antigen receptor can comprise an scFv targeting the MUC1 alpha subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the scFv can comprise the amino acid sequence set forth in SEQ ID NO: 213.

[0297] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting MUC1 beta subunit and MUC1 alpha subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the antigen binding domain targeting MUC1 beta subunit and MUC1 alpha subunit comprising the aforementioned antigen binding protein targeting MUC1 beta subunit and the aforementioned antigen binding protein targeting MUC1 alpha subunit. For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining region HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO: 190; the second antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining region HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 205, the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 206.For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-31; the second antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 205, the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 206. For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-31, the LCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 77-83, the LCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 84-89, the LCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 90-96; the second antigen binding protein can comprise an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 205, the HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 206, the VL comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 87, the LCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 211.For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 147-159 and a light chain variable region VL, the second antigen binding protein can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in SEQ ID NO: 203 and a light chain variable region VL. For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 147-159 and a light chain variable region VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 163-175, the second antigen binding protein can comprise an antibody heavy chain variable region VH comprising an amino acid sequence set forth in SEQ ID NO: 203 and a light chain variable region VL comprising an amino acid sequence set forth in SEQ ID NO: 210.

[0298] For example, the chimeric antigen receptor can comprise an scFv targeting a MUC1 β subunit, an scFv targeting a MUC1 α subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the scFv targeting a MUC1 β subunit can comprise an amino acid sequence set forth in any one of SEQ ID NOs: 142-146 or SEQ ID NOs: 194-201, the scFv targeting a MUC1 α subunit can comprise an amino acid sequence set forth in SEQ ID NO: 213.

[0299] For example, the chimeric antigen receptor can comprise an antigen binding domain targeting MUC1 β subunit and MUC1 α subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the antigen binding domain can comprise a first antigen binding protein and a second antigen binding protein, the first antigen binding protein can comprise a VHH comprising a heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 13, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22, and the second antigen binding protein can comprise an antibody heavy chain variable region VH comprising a heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 204, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 205, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 206, and a light chain variable region VL comprising a light chain complementarity determining region LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 80, a LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 87, and a LCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 211.

[0300] For example, the chimeric antigen receptor can comprise a VHH targeting MUC1 β subunit, an scFv targeting MUC1 α subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the VHH targeting MUC1 β subunit can comprise an amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO: 160, and the scFv targeting MUC1 α subunit can comprise an amino acid sequence set forth in SEQ ID NO: 213.

[0301] In the present application, the chimeric antigen receptor can further comprise a signal peptide. For example, the signal peptide can be a CD8 signal peptide. For example, the amino acid sequence of the CD8 signal peptide can be set forth in SEQ ID NO: 214.

[0302] MUC1 α subunit as an activator

[0303] The therapeutic potential of CAR-T therapy in the field of hematological tumors is undisputed, but the research in solid tumors is not satisfactory, one of the main reasons is that CAR-T cannot effectively expand in vivo, leading to decreased efficacy. For CAR-T targeting the abnormal glycosylation site of MUC1 a subunit, the laboratory found that free CA153 in the blood of tumor patients can effectively promote the low-level expansion of CAR-T cells and maintain the activated state, which helps the killing effect of CAR-T in vivo.

[0304] In another aspect, the present application provides an activator comprising a MUC1 a subunit, the activator is used to activate and expand CAR-T cells targeting the MUC1 a subunit. In the present application, the CAR-T can target the tandem repeat sequence of the MUC1 a subunit. In the present application, the CAR-T cell can trigger the multimerization of the chimeric antigen receptor by binding to the tandem repeat sequence of the MUC1 a subunit. In the present application, the CAR-T cell can have at least three chimeric antigen receptors on the same cell simultaneously binding to the tandem repeat sequence of the MUC1 a subunit. In the present application, the chimeric antigen receptor of the CAR-T cell can trigger the activation and expansion of the CAR-T after binding to the tandem repeat sequence of the MUC1 a subunit.

[0305] In another aspect, the present application provides the use of a MUC1 a subunit in the preparation of the activator.

[0306] Modified immune cells

[0307] In another aspect, the present application provides a modified immune cell, wherein the immune cell can comprise the aforementioned chimeric antigen receptor.

[0308] In the present application, the immune cell can be a T cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a γδ T cell, and / or a DNT cell. For example, the immune cell can be an immune effector cell. For example, the immune cell can be a T cell. For example, the immune cell can be a mixture, which can comprise different immune cell species, for example, the mixture can comprise one or more immune cells.

[0309] In the present application, the immune cell can comprise and / or express one or more antigen recognition receptors. In the present application, the immune cell can comprise and / or express one or more antigen recognition receptors. In the present application, the immune cell can comprise and / or express one or more chimeric antigen receptors. In the present application, the immune cell can comprise and / or express one or more chimeric antigen receptors. For example, the immune cell can comprise the aforementioned chimeric antigen receptor targeting the MUC1 β subunit and the aforementioned chimeric antigen receptor targeting the MUC1 a subunit.

[0310] In another aspect, the present application provides a modified immune cell expressing:

[0311] a) a first chimeric antigen receptor, the first chimeric antigen receptor comprising a transmembrane domain, an intracellular signaling domain, and a first binding element, wherein the first binding element specifically interacts with a first target antigen that comprises a tandem repeat sequence, and the first binding element binds to the tandem repeat sequence; and

[0312] b) a second chimeric antigen receptor, the second chimeric antigen receptor comprising a transmembrane domain, an intracellular signaling domain, and a second binding element, wherein the second binding element specifically interacts with a second target antigen.

[0313] In the present application, the first chimeric antigen receptor and / or the second chimeric antigen receptor can further comprise a costimulatory domain.

[0314] In the present application, the first binding element and / or the second binding element can be the aforementioned antigen binding protein. For example, the first binding element can bind to a MUC1 a subunit. For example, the second binding element can bind to a MUC1 b subunit. For example, the first binding element can be the aforementioned antigen binding protein targeting the MUC1 a subunit, and the second binding element can be the aforementioned antigen binding protein targeting the MUC1 b subunit.

[0315] In the present application, the first chimeric antigen receptor can trigger multimerization of the first chimeric antigen receptor by binding to the tandem repeat sequence of the first target antigen. In the present application, the first target antigen can be a MUC1 a subunit. In the present application, the MUC1 a subunit has a tandem repeat sequence. For example, the first chimeric antigen receptor triggers multimerization of the first chimeric antigen receptor by binding to the tandem repeat sequence of the MUC1 a subunit.

[0316] In the present application, the modified immune cell can have at least three first chimeric antigen receptors on the same cell simultaneously binding to the tandem repeat sequence of the first target antigen. For example, the modified immune cell can have at least three first chimeric antigen receptors on the same cell simultaneously binding to the tandem repeat sequence of the MUC1 a subunit.

[0317] In the present application, the first chimeric antigen receptor binding to the tandem repeat sequence of the first target antigen can trigger activation and expansion of the modified immune cell. For example, the first chimeric antigen receptor binding to the tandem repeat sequence of the MUC1 a subunit can trigger activation and expansion of the modified immune cell.

[0318] In the present application, the first target antigen can be present in serum and / or body fluid. For example, the first target antigen can be present in serum. For example, the first target antigen can be present in body fluid. In the present application, the body fluid can be ascites. For example, the first target antigen can be present in ascites. In the present application, the concentration of the first target antigen in serum and / or body fluid can be 0-358 IU / ml. In the present application, the concentration of the first target antigen in serum and / or body fluid can be 12-250 IU / ml.

[0319] In the present application, the first target antigen can be a tumor antigen. For example, the tumor antigen can be a tumor associated antigen. For example, the tumor antigen can be a tumor specific antigen. For example, the first target antigen can be a shed MUC1 a subunit (CA153). In the present application, the first chimeric antigen receptor can bind to the tandem repeat sequence of the shed MUC1 a subunit to trigger the first chimeric antigen receptor multimerization. In the present application, the modified immune cell can have at least three first chimeric antigen receptors simultaneously bind to the tandem repeat sequence of the shed MUC1 a subunit on the same cell. In the present application, the first chimeric antigen receptor can trigger the activation and expansion of the modified immune cell after binding to the tandem repeat sequence of the shed MUC1 a subunit.

[0320] In another aspect, the present application provides a modified immune cell expressing a tandem chimeric antigen receptor (CAR) comprising a transmembrane domain, an intracellular signaling domain, and a tandem binding element, wherein the tandem binding element interacts with a first target antigen comprising a tandem repeat sequence and specifically interacts with a second target antigen, and the tandem binding element binds to the tandem repeat sequence.

[0321] In the present application, the tandem chimeric antigen receptor can further comprise a costimulatory domain.

[0322] In the present application, the tandem chimeric antigen receptor can be the aforementioned antigen binding protein. For example, the tandem chimeric antigen receptor can be the aforementioned chimeric antigen receptor targeting MUC1 b subunit and MUC1 a subunit.

[0323] In the present application, the tandem chimeric antigen receptor comprises a transmembrane domain, an intracellular signaling domain, a costimulatory domain, and a tandem binding element, wherein the tandem binding element specifically interacts with a first target antigen comprising a tandem repeat sequence and a second target antigen, and the tandem binding element binds to the tandem repeat sequence.

[0324] In the present application, the tandem binding element can comprise the aforementioned antigen binding protein. For example, the tandem binding element can comprise the aforementioned antigen binding protein targeting the MUC1 a subunit and the aforementioned antigen binding protein targeting the MUC1 β subunit. In the present application, the tandem binding element can be a fusion protein.

[0325] In the present application, the tandem binding element can comprise a first single chain antibody and a second single chain antibody, wherein the first single chain antibody specifically interacts with a first target antigen and the second single chain antibody specifically interacts with a second target antigen. In the present application, the first single chain antibody and the second single chain antibody can be directly or indirectly linked. For example, the first single chain antibody and the second single chain antibody can be indirectly linked by a linker.

[0326] In the present application, the tandem chimeric antigen receptor binding the first target antigen tandem repeat sequence can trigger the multimerization of the tandem chimeric antigen receptor. In the present application, the first target antigen can be the MUC1 a subunit. For example, the tandem chimeric antigen receptor triggers the multimerization of the tandem chimeric antigen receptor by binding the tandem repeat sequence of the MUC1 a subunit.

[0327] In the present application, the modified immune cell can have at least three tandem chimeric antigen receptors simultaneously bind to the first target antigen tandem repeat sequence on the same cell. For example, the modified immune cell can have at least three tandem chimeric antigen receptors simultaneously bind to the tandem repeat sequence of the MUC1 a subunit on the same cell.

[0328] In the present application, the tandem chimeric antigen receptor binding the first target antigen tandem repeat sequence can trigger the activation and expansion of the modified immune cell. For example, the tandem chimeric antigen receptor binding the tandem repeat sequence of the MUC1 a subunit can trigger the activation and expansion of the modified immune cell.

[0329] In the present application, the tandem chimeric antigen receptor binding the CA153 tandem repeat sequence can trigger the multimerization of the tandem chimeric antigen receptor. In the present application, the modified immune cell can have at least three tandem chimeric antigen receptors simultaneously bind to the CA153 tandem repeat sequence on the same cell.

[0330] In the present application, the tandem chimeric antigen receptor binding the CA153 tandem repeat sequence can trigger the activation and expansion of the modified immune cell.

[0331] In the present application, the CA153 can exist in serum and / or body fluid. For example, the CA153 can exist in serum. For example, the CA153 can exist in body fluid. In the present application, the body fluid can be ascites. For example, the CA153 can exist in ascites. In the present application, the CA153 concentration in serum and / or body fluid can be 0-358 IU / ml. In the present application, the CA153 concentration in serum and / or body fluid can be 12-250 IU / ml.

[0332] In the present application, the first target antigen tandem repeat sequence amino acid repeat unit can repeat 20-125 times. In the present application, the first target antigen can be CA153, and the CA153 tandem repeat sequence has an amino acid repeat unit as shown in SEQ ID NO: 221. In the present application, the CA153 tandem repeat sequence amino acid repeat unit can repeat 20-125 times.

[0333] In the present application, the modified immune cells can bind to the exfoliated antigen in the serum and / or body fluid of the subject. For example, the modified immune cells can bind to the exfoliated antigen in the serum of the subject. For example, the modified immune cells can bind to the exfoliated antigen in the body fluid of the subject. In the present application, the body fluid can be ascites. For example, the modified immune cells can bind to the exfoliated antigen in the ascites of the subject.

[0334] In the present application, the subject can have a tumor. For example, the tumor can be a solid tumor. For example, the tumor can be breast cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, gastric cancer, myeloma, liver cancer and / or bile duct cancer.

[0335] In the present application, the exfoliated antigen concentration in the serum and / or body fluid of the subject can be 0-358 IU / ml. For example, the first target antigen concentration in the serum and / or body fluid of the subject can be 12-250 IU / ml.

[0336] In the present application, the second target antigen can be located on the cell surface. In the present application, the second target antigen can be a tumor antigen. For example, the tumor antigen can be a tumor-associated antigen. For example, the tumor antigen is MUC1 β subunit.

[0337] In the present application, the modified immune cells can specifically interact with the CA153 tandem repeat sequence and the MUC1 β subunit.

[0338] In the present application, the transmembrane domain, the intracellular signaling domain, the costimulatory domain can be as described above.

[0339] In the present application, the modified immune cell can comprise:

[0340] a) a first chimeric antigen receptor comprising a CD8 signal peptide, a first binding element targeting a shed MUC1 alpha subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain;

[0341] b) a second chimeric antigen receptor comprising a CD8 signal peptide, a second binding element targeting a MUC1 beta subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0342] In the present application, the modified immune cell can comprise a tandem chimeric antigen receptor, wherein the tandem chimeric antigen receptor comprises: a CD8 signal peptide, a first single-chain antibody targeting a shed MUC1 alpha subunit, a (GGGGS)4 linker, a second single-chain antibody targeting a MUC1 beta subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0343] In the present application, the each part of the amino acid sequence can be directly or indirectly connected. For example, the each part of the amino acid sequence can be directly connected. For example, the each part of the amino acid sequence can be indirectly connected. For example, the each part of the amino acid sequence can be indirectly connected by a linker.

[0344] Immunoconjugate

[0345] In another aspect, the present application also provides an immunoconjugate, wherein the immunoconjugate can comprise the antigen binding protein. In the present application, the immunoconjugate can specifically bind to a MUC1 beta subunit and / or a MUC1 alpha subunit.

[0346] In the present application, the immunoconjugate can comprise an antigen binding protein, a linker, and a payload. In the present application, the each part of the immunoconjugate is directly or indirectly connected. For example, the each part of the immunoconjugate is indirectly connected. For example, the each part of the immunoconjugate is connected by a linker.

[0347] In the present application, the payload can be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid.

[0348] In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a radioconjugate (RDC).

[0349] In the present application, the antibody drug conjugate can comprise the antigen binding protein, a linker, and a cytotoxic drug. In the present application, the cytotoxic drug can be a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In the present application, the chemotherapeutic drug can be vinblastine, doxorubicin, or a derivative thereof. In the present application, the tubulin inhibitor can be auristatin, eribulin, maytansine, or a derivative thereof. In the present application, the DNA damaging agent can be a calicheamicin, a duocarmycin, an anthramycin derivative PBD, or a derivative thereof. In the present application, the topoisomerase I inhibitor can be camptothecin, topotecan, irinotecan, or a derivative thereof.

[0350] In the present application, the PROTAC-antibody conjugate can comprise the antigen binding protein, a linker, and a PROTAC. In the present application, the PROTAC can comprise a target protein ligand, a linker, an E3 ligase ligand. In the present application, the E3 ligase ligand can be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0351] In the present application, the targeted nuclear agent can comprise the antigen binding protein, a linker, and a radioisotope. In the present application, the radioisotope can be iodine-131, radium-223, thallium-201, or arsenic-211.

[0352] Nucleic acid molecules, vectors, and cells

[0353] In another aspect, the present application also provides an isolated nucleic acid molecule, which can encode the antigen binding protein and / or the chimeric antigen receptor.

[0354] In the present application, the nucleic acid molecule can further comprise a sequence encoding a signal peptide. In the present application, the nucleic acid molecule can be produced or synthesized by (i) amplification in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by recombinant cloning, (iii) purified, such as fractionated by enzyme digestion and gel electrophoresis, or (iv) synthesized, such as by chemical synthesis.

[0355] In the present application, the nucleic acid molecule can be DNA and / or RNA. In the present application, the nucleic acid molecule can be an artificially synthesized nucleic acid analogue. In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0356] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0357] In the present application, the vector can comprise one or more of the nucleic acid molecules. In the present application, the vector can comprise one or more of the nucleic acid molecules.

[0358] In the present application, the vector can be an expression vector or a cloning vector. In the present application, the vector can be a viral vector or a non-viral vector. In the present application, the vector can be a viral vector, a plasmid vector, a phage vector, or other vectors commonly used in, for example, genetic engineering. For example, the viral vector can be an adenovirus, an adeno-associated virus, a retrovirus (including lentivirus). In the present application, the vector can be a fusion vector or a non-fusion vector.

[0359] In the present application, the vector can further comprise other genes. For example, the other genes can be marker genes.

[0360] In the present application, the vector can contain various elements that control expression. For example, the vector can include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector can also contain a replication initiation site. For example, the vector can include components that assist in entry into a cell. To allow the nucleic acid molecule to replicate in the vector, the 5' end and the 3' end of the nucleic acid molecule can also comprise long terminal repeat sequences.

[0361] On the other hand, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0362] In the present application, the cell can include progeny of a single cell. The progeny can not necessarily be identical to the original parent cell (in the morphology of the total DNA complement or in the genome) due to natural, accidental, or intentional mutations.

[0363] In the present application, the cell can be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, or a HEK293A cell, or other eukaryotic cells such as fungal or yeast cells, etc.

[0364] In the present application, the cell can comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector can comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0365] In the present application, the vector can be introduced into the cell by a method known in the art. For example, the method can be electroporation, Lipofectine transfection, or Lipofectamin transfection.

[0366] In another aspect, the present application provides a method of preparing the aforementioned antigen binding protein, the method comprising culturing the cell under conditions such that the antigen binding protein is expressed.

[0367] Pharmaceutical composition

[0368] In another aspect, the present application also provides a pharmaceutical composition, which can comprise the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0369] In the present application, the pharmaceutical composition can comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations employed. The pharmaceutical composition of the present application can include liquid, frozen and lyophilized compositions.

[0370] In the present application, the pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents which are compatible with pharmaceutical administration, generally safe, non-toxic and neither biologically nor otherwise undesirable.

[0371] In the present application, the pharmaceutical composition can comprise parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, administration of the pharmaceutical composition can be performed in different ways, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0372] Pharmaceutical combination

[0373] In another aspect, the present application provides a pharmaceutical combination comprising the antigen binding protein, the pharmaceutical combination can further comprise one or more active ingredients other than the antigen binding protein.

[0374] For example, the pharmaceutical combination can further comprise a substance related to an immune response. For example, the pharmaceutical combination can further comprise a drug related to an immune response.

[0375] In another aspect, the present application provides a pharmaceutical combination comprising the antigen binding protein, the nucleic acid molecule and / or the cell and a therapeutic agent. In the present application, the therapeutic agent can be selected from one or more of the following group: an antitumor drug, a chemotherapeutic agent, a radioisotope or an immune checkpoint inhibitor.

[0376] On the other hand, the present application also provides a scheme for combining the antigen-binding protein with one or more other active ingredients. For example, the antigen-binding protein is combined with one or more other therapeutic agents. In the present application, the drug combination can be administered separately, simultaneously, or sequentially. In the present application, the drug combination can be administered in the same or different doses or administration routes. For example, the active ingredients in the drug combination are administered to the patient as separate entities in the same / different doses and administration routes. In the present application, the ingredients in the drug combination can be administered to the patient simultaneously in the form of a single entity or dose. For example, the ingredients in the drug combination are administered to the patient simultaneously, jointly, or sequentially as separate entities. In the present application, the specific administration route can be determined according to the type of active ingredient, and the specific dosage can be adjusted according to the severity of the subject's condition, the subject's physical condition, etc.

[0377] In the present application, the different active ingredients in the pharmaceutical combination may be mixed or placed separately. For example, the active ingredients may be placed in the same container. For example, the active ingredients may be placed in different containers.

[0378] Preparation method

[0379] In another aspect, the present application provides a method for preparing the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition. For example, the method may include culturing the cell under conditions that allow the antigen-binding protein and / or the chimeric antigen receptor to be expressed. For example, the method may include introducing the vector into the immune cell.

[0380] use

[0381] On the other hand, the present application also provides the use of the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition in the preparation of a drug, which can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0382] On the other hand, the present application also provides a method for preventing, diagnosing and / or treating diseases and / or conditions, which may include administering the antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition to a subject in need.

[0383] On the other hand, the present application also provides antigen binding proteins, chimeric antigen receptors, modified immune cells, immunoconjugates, nucleic acid molecules, vectors, cells and / or pharmaceutical compositions, which can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0384] In the present application, the disease and / or disorder can be a MUC1 beta subunit and / or MUC1 alpha subunit related disease and / or disorder.

[0385] In the present application, the disease and / or disorder can be a tumor. In the present application, the tumor can be a solid tumor and / or a blood tumor. In the present application, the tumor can be a MUC1 beta subunit and / or MUC1 alpha positive tumor. In the present application, the tumor can be a MUC1 beta subunit and / or MUC1 alpha positive tumor of epithelial origin. In the present application, the tumor can be a breast cancer, a prostate cancer, a colorectal cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, a gastric cancer, a myeloma, a liver cancer and / or a cholangiocarcinoma.

[0386] In the present application, the prevention, diagnosis and / or treatment can be preventing the onset of the disease, slowing or reversing the course of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0387] In another aspect, the present application provides the use of the antigen binding protein according to the application for the manufacture of a diagnostic agent for the diagnosis of a disease and / or disorder associated with the expression of MUC1 beta subunit and / or MUC1 alpha.

[0388] In another aspect, the present application provides the use of the antigen binding protein according to the application for the manufacture of a diagnostic agent for the diagnosis of a disease and / or disorder associated with the expression of MUC1 beta subunit and / or MUC1 alpha.

[0389] In the present application, the diagnostic agent can be used alone or in combination with an instrument, appliance, device or system. The diagnostic agent can be used for in vitro detection of human samples (e.g. various body fluids, cells, tissue samples, etc.) in the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases.

[0390] In the present application, the diagnostic agent can be selected from the group consisting of reagents, kits, calibrators and quality controls.

[0391] In the present application, the method of in vitro detection can be selected from the group consisting of Western Blot, ELISA and immunohistochemistry.

[0392] For example, the reagent can comprise a reagent capable of measuring the amount of expression of the MUC1 beta subunit and / or MUC1 alpha.

[0393] For example, the reagents can be selected from the group of reagents to perform Western Blot, reagents to perform ELISA, and reagents to perform immunohistochemistry.

[0394] In another aspect, the present application also provides a detection kit, which can comprise the antigen binding protein, for detecting the presence and / or amount of MUC1 β subunit and / or MUC1 α in a sample or subject. For example, the detection kit can be used for preventing, diagnosing and / or treating diseases and / or disorders.

[0395] For example, the present application relates to an immunodetection kit for use in immunodetection methods such as ELISA, immunohistochemistry, Western Blot, flow cytometry, etc. using the antibodies of the present application.

[0396] In the present application, the antigen binding protein of the present application is included in the kit for detecting MUC1 β subunit and / or MUC1 α related cancer cells, in particular, the immunodetection kit will comprise the antibody of the present application as a first antibody that binds to MUC1 β subunit and / or MUC1 α in a suitable container means, and optionally an immunodetection reagent.

[0397] For example, the antibody can be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well.

[0398] In the present application, the immunodetection reagents in the kit can be in any of a variety of formats, including those detectable labels bound or linked to a given antibody. Detectable labels bound or linked to secondary binding ligands can also be included. Exemplary secondary ligands are those secondary antibodies having binding affinity for the first antibody.

[0399] In the present application, other immunodetection reagents suitable for use in the kits of the present application include two-component reagents comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody linked to a detectable label. As noted above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present application.

[0400] In another aspect, the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition of the present application are used for the manufacture of a diagnostic detection kit.

[0401] In the present application, the kit can further comprise a composition of MUC1 beta subunit and / or MUC1 alpha, in appropriate aliquots, whether labeled or not, which can be used to prepare a standard curve for the detection assay. The kit can contain the antibody-label conjugate in a fully conjugated form, in an intermediate form, or separate parts to be conjugated by the user of the kit. The components of the kit can be packaged in an aqueous medium or in lyophilized form.

[0402] In the present application, the container means of the kit will generally comprise at least one vial, test tube, flask, bottle, syringe, or other container means, into which the antibody or preferably the antibody in appropriate aliquots can be placed. The kit of the present application will also typically include a container means for containing the antibody, antigen and any other reagents containers in sealed form, ready for use.

[0403] In another aspect, the present application also provides a method of detecting the presence and / or amount of MUC1 beta subunit and / or MUC1 alpha, which can comprise the use of said antigen binding protein.

[0404] In another aspect, the present application provides a method of diagnosing a disease and / or disorder associated with the expression of MUC1 beta subunit and / or MUC1 alpha in a subject, said method comprising: contacting a sample derived from said subject with said antigen binding protein, and determining the presence and / or amount of a substance capable of specifically binding to said antigen binding protein in said sample.

[0405] In another aspect, the present application provides a method of detecting MUC1 beta subunit and / or MUC1 alpha in a sample or a subject, said method comprising administering said antigen binding protein. In the present application, said administration can be performed by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0406] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the antigen binding proteins, the methods of preparation and uses of the present application, and are not intended to limit the scope of the present application.

[0407] Examples

[0408] Example 1. panning and binding validation of VHH format antibodies targeting MUC1 beta subunit

[0409] 1.1 Generation of VHH format antibody library and sequence panning

[0410] This example synthesizes MUC1 β subunit extracellular ECD short peptide (SEQ ID NO: 140), which is coupled with KLH, and then immunizes alpacas and builds a library by a company in accordance with known protocols in the art. The first immunization dose is 0.5 mg of protein, and the last three immunizations use 0.25 mg of protein. After the third and fourth immunizations, 50 mL of peripheral blood is collected for standby, and a small amount of serum is separated for ELISA detection of immune effect. The final third and fourth immunization titers are both above 1:64000 (coating antigen 2 μg / mL, OD positive value greater than 2.0), meeting the requirements for library construction. PBMC is separated and total RNA of the third and fourth immunized PBMC is extracted using RNAiso Plus reagent, and the extracted RNA is reverse transcribed into cDNA using a first strand cDNA synthesis kit (PrimeScript TM II 1st Strand cDNA Synthesis Kit, 6210A, Takara). In accordance with known protocols in the art, the variable region (VHH) of the heavy chain antibody is amplified by two rounds of nested PCR, the target fragment is recovered by gel recovery, and the restriction enzyme SfiI is used to clone it into the phage display vector pComb3XSS. After desalting the plasmid, it is electroporated into electrocompetent E. coli TG1 to construct a phage display nanobody library.

[0411] In addition, 296 non-redundant nanobodies and their structures in the PDB database (Protein Data Bank) are aligned to determine the CDR mutation strategy. The nanobody Caplacizumab, which has been marketed, is selected as the scaffold, and the nucleotide sequence of Caplacizumab is synthesized, and then cloned into the HP153 phage vector. Then single-stranded DNA of HP153 is extracted, and the CDRs of the nanobody are mutated using the Kunkel method to obtain double-stranded DNA. The double-stranded DNA is electroporated into competent E. coli SS320 pre-infected with M13KO7 helper phage, and after overnight culture, the phage supernatant is collected to construct an artificially synthesized nanobody library.

[0412] Finally, magnetic bead liquid selection obtains a MUC1 β subunit monoclonal sequence 1A1 from the VHH form phage immune library, and a candidate sequence 1A2 from the artificially synthesized nanobody library.

[0413] The obtained antibody nucleotide sequence is subcloned into the eukaryotic expression vector pcDNA3.4, and then transfected into Expi 293 cells to culture and express for 5 days to prepare the antibody. The supernatant is collected and purified by protein A (Genscript, Cat. No. L00695-80).

[0414] The VHH amino acid sequence of 1A1 is shown as SEQ ID NO: 160, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 13, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 22.

[0415] The VHH amino acid sequence of 1A2 is shown as SEQ ID NO: 161, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 14, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 23.

[0416] 1.2 Specific binding verification of VHH format candidate antibodies

[0417] The specific binding activity of MUC1 β subunit mAbs 1A1 and 1A2 was identified by flow cytometry. HCT116 empty cells and MUC1 β subunit overexpressed HCT116 cells (HCT116-MUC1 β) were counted, the cells were resuspended with flow buffer and adjusted to 1 × 10 6 / ml, and 30 μl / well of each of the two cells were added to a V-bottom 96-well plate. 30 μl / well of the candidate antibody and the control antibody were added, wherein 1A1 and the control antibody H44 (SEQ ID NO: 162, Minerva Biotechnologies, against MUC1 β subunit) were started at a working concentration of 25 μg / ml, diluted with flow buffer at a gradient of 2 times, 9 gradients were formed, and PBS negative controls were set for each antibody. 1A2 and the control antibody H44 were started at a working concentration of 25 μg / ml, diluted with flow buffer at a gradient of 2 times, 11 gradients were formed, and PBS negative controls were set for each antibody. Incubate at 4°C for 1 hour, wash once with flow buffer, add 30 μl / well of anti-human IgG-Fc secondary antibody (APC), incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, and add 25 μl / well of flow buffer to the machine. The raw data were plotted and calculated using GraphPad 8.0 software.

[0418] The binding activity results with target cells are shown in Figures 1 and 2. The VHHs 1A1 and 1A2 of the present application can detect a certain MFI value on MUC1 β expressing target cells, indicating that the VHHs can bind to the cell surface MUC1 β, and the antigen binding activity of 1A1 and 1A2 is comparable to or higher than that of the control antibody H44 at some antibody concentrations, while hardly binding to cells not expressing MUC1 β. The experimental results show that the VHHs described in the present application can specifically bind to the MUC1 β subunit and have high affinity.

[0419] Generation and binding verification of camelid MUC1 β subunit antibody

[0420] 2.1 Generation and sequence panning of camelid scFv format antibody library

[0421] Based on the total RNA of three and four immunized PBMCs extracted in Example 1.1, the first strand cDNA kit (PrimeScript TM II 1st Strand cDNA Synthesis Kit, 6210A, Takara) was used to reverse transcribe it into cDNA. Under the known scheme in the art, the antibody VH, VK, VL variable regions were amplified by two rounds of nested PCR, the target fragments were gel recovered, and the scFv (VH+VK, VH+VL) structure was constructed by one round of overlap, in which the heavy and light chain variable regions were connected by a (G4S)3 linker, the target fragments were gel recovered, and the restriction enzyme Sfil was used to clone them into the phage display vector pComb3XSS. After desalting the plasmid, it was electroporated into electrocompetent E. coli TG1 to construct the phage display nanobody library.

[0422] Finally, three MUC1 β subunit monoclonal sequences targeting scFv format phage immune library from camelids, namely 1A3, 1A4 and 1A5, were obtained by magnetic bead liquid panning.

[0423] The scFv amino acid sequence of 1A3 is shown as SEQ ID NO: 194, the VH amino acid sequence is shown as SEQ ID NO: 147, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 24; the VL amino acid sequence is shown as SEQ ID NO: 163, the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 84, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 90.

[0424] The scFv amino acid sequence of 1A4 is shown as SEQ ID NO: 195, the VH amino acid sequence is shown as SEQ ID NO: 148, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 25; the VL amino acid sequence is shown as SEQ ID NO: 164, the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 91.

[0425] The scFv amino acid sequence of 1A5 is shown as SEQ ID NO: 196, the VH amino acid sequence is shown as SEQ ID NO: 150, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 26; the VL amino acid sequence is shown as SEQ ID NO: 166, the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 92.

[0426] Specific binding verification of camelid scFv format candidate antibodies

[0427] The specific binding activity of three candidate MUC1 β subunit antibodies and the positive control antibody H44 (scFv format, same below) was identified by flow cytometry. HCT116 empty cells and MUC1 β subunit overexpressing HCT116 cells (HCT116-MUC1 β) were counted, resuspended with flow buffer and adjusted to 1 × 10 6 / ml, and 30 μl / well of each cell was added to a V-bottom 96-well plate. 30 μl / well of candidate antibodies and positive controls were added at a starting concentration of 50 μg / ml, diluted with flow buffer at a 2-fold ratio gradient to form 11 gradients, and PBS blank control wells were set. Incubate at 4°C for 1 hour, wash once with flow buffer, add 30 μl / well of anti-human IgG Fc secondary antibody (APC), incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, and add 25 μl / well of flow buffer to the machine. Replace the original data into the GraphPad 8.0 software for plotting and calculation.

[0428] The results of the binding activity with target cells are shown in Figure 3. The scFv forms 1A3, 1A4 and 1A5 antibodies of the present application can all detect a certain MFI value on target cells expressing MUC1 β, indicating that the scFv form antibodies can all bind to the cell surface MUC1 β, and the antigen binding activity of the scFv form antibodies of the present application is higher than that of the control antibody H44, and almost no binding on cells not expressing MUC1 β. The experimental results show that the scFv form antibodies described in the present application can specifically bind to MUC1 β subunit, and have high affinity.

[0429] The binding activity of MUC1 β subunit antibodies 1A3, 1A4, 1A5 and positive control antibody H44 with embryonic stem cells H1 naturally expressing MUC1 β subunit was identified by flow cytometry. H1 cells were counted, resuspended with flow buffer and adjusted to 1×10 6 / ml, 30 μl / well was added to a V-bottom 96-well plate. 30 μl / well of the antibody to be verified and the positive control antibody was added, which was started at a working concentration of 50 μg / ml, diluted by 2 times ratio gradient, forming 7 gradients, and PBS negative control was set for each antibody. Incubate at 4°C for 1 hour, wash once with flow buffer, add 30 μl / well of anti-human IgG-Fc secondary antibody (APC), incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, add 25 μl / well of flow buffer to the machine. The original data is substituted into the GraphPad 8.0 software for plotting and calculation.

[0430] The results of the binding activity with embryonic stem cells are shown in Figure 4. With the increase of the concentration of the scFv form antibodies described in the present application, no significant increase in average fluorescence intensity was observed. The experimental results show that the scFv form antibodies described in the present application do not bind to embryonic stem cells.

[0431] Example 3 Generation and binding verification of murine MUC1 β subunit antibodies

[0432] 3.1 Selection of murine candidate chimeric antibody sequences

[0433] The present example synthesizes MUC1 β subunit extracellular ECD short peptide (SEQ ID NO: 140), which is coupled with KLH, and then immunizes Balb / c mice for four times by entrusting Aikangde Biotech (Suzhou) Co., Ltd. under the scheme known in the art. Each immunization uses 0.1 mg of coupled antibody. After the third and fourth immunization, a small amount of separated serum is collected, and the immune effect is detected by ELISA. The final fourth immunization titer is more than 1:125000 (coating antigen 1 μg / mL, OD positive value greater than 2.0), which meets the requirements. The mouse hybridoma technology is used to obtain MUC1 β subunit positive monoclonal cells by flow cytometry screening and subcloning, and IgG type MUC1 β subunit mouse monoclonal antibody is obtained by in vitro culture. The heavy chain variable region (VH) and light chain variable region (VL) sequences are obtained by sequencing using the degenerate primer amplification method, and are respectively combined with the corresponding light and heavy chain constant regions CH to construct chimeric antibodies 1A6, 1A7, 1A8, 1A9 and 1A10.

[0434] The heavy chain amino acid sequence of chimeric antibody 1A6 is shown as SEQ ID NO: 181, the VH amino acid sequence is shown as SEQ ID NO: 152, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 27; the light chain amino acid sequence is shown as SEQ ID NO: 176, the VL amino acid sequence is shown as SEQ ID NO: 168, the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 93.

[0435] The heavy chain amino acid sequence of chimeric antibody 1A7 is shown as SEQ ID NO: 182, the VH amino acid sequence is shown as SEQ ID NO: 154, the HCDR1 comprises the amino acid sequence shown as SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 28; the light chain amino acid sequence is shown as SEQ ID NO: 177, the VL amino acid sequence is shown as SEQ ID NO: 170, the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 94.

[0436] The heavy chain amino acid sequence of chimeric antibody 1A8 is set forth in SEQ ID NO: 183, the VH amino acid sequence is set forth in SEQ ID NO: 155, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; the light chain amino acid sequence is set forth in SEQ ID NO: 178, the VL amino acid sequence is set forth in SEQ ID NO: 171, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94.

[0437] The heavy chain amino acid sequence of chimeric antibody 1A9 is set forth in SEQ ID NO: 184, the VH amino acid sequence is set forth in SEQ ID NO: 157, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; the light chain amino acid sequence is set forth in SEQ ID NO: 179, the VL amino acid sequence is set forth in SEQ ID NO: 173, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 89, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95.

[0438] The heavy chain amino acid sequence of chimeric antibody 1A10 is set forth in SEQ ID NO: 185, the VH amino acid sequence is set forth in SEQ ID NO: 159, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31; the light chain amino acid sequence is set forth in SEQ ID NO: 180, the VL amino acid sequence is set forth in SEQ ID NO: 175, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0439] 3.2 Specific binding verification of murine candidate chimeric antibodies

[0440] The specific binding activity of the MUC1 β subunit chimeric antibody and the positive control antibody H44 was identified by flow cytometry. HCT116 empty cells and MUC1 β subunit overexpressing HCT116 cells (HCT116-MUC1 β) were counted, resuspended with flow buffer and adjusted to 1 x 10 6 / ml, and 30 μl / well of each cell was added to a V-bottom 96-well plate. 30 μl / well of the candidate antibody and the positive control antibody was added, which was started at a working concentration of 50 μg / ml and diluted with flow buffer at a 2-fold ratio gradient to form 7 gradients, and PBS blank control wells were set. Incubate at 4°C for 1 hour, wash once with flow buffer, add 30 μl / well of anti-human IgG Fc secondary antibody (APC), incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, and add 25 μl / well of flow buffer to the machine. The raw data was imported into GraphPad 8.0 software for plotting and calculation.

[0441] The binding activity results with target cells are shown in Figure 5. The chimeric antibodies 1A6, 1A7, 1A8, 1A9 and 1A10 of the present application can detect a certain MFI value on MUC1 β expressing target cells, indicating that the chimeric antibodies can bind to the cell surface MUC1 β, and the antigen binding activity of the chimeric antibodies of the present application is higher than that of the control antibody H44, and almost no binding is observed on cells not expressing MUC1 β. The experimental results show that the chimeric antibodies described in the present application can specifically bind to the MUC1 β subunit and have high affinity.

[0442] The binding activity of each antibody of the MUC1 β subunit and the control antibody H44 with the embryonic stem cells H1 naturally expressing the MUC1 β subunit was identified by flow cytometry. H1 cells were counted, resuspended with flow buffer and adjusted to 1 x 10 6 / ml, and 30 μl / well was added to a V-bottom 96-well plate. 30 μl / well of the candidate antibody and the positive control antibody was added, which was started at a working concentration of 50 μg / ml and diluted with flow buffer at a 2-fold ratio gradient to form 7 gradients, and PBS negative controls were set for each antibody. Incubate at 4°C for 1 hour, wash once with flow buffer, add 30 μl / well of anti-human IgG-Fc secondary antibody (APC), incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, and add 25 μl / well of flow buffer to the machine. The raw data was imported into GraphPad 8.0 software for plotting and calculation.

[0443] The binding activity results with embryonic stem cells are shown in Figure 6. With the increase of the concentration of the chimeric antibodies described in the present application, no significant increase in the average fluorescence intensity was observed. The experimental results show that the candidate chimeric antibodies described in the present application do not bind to embryonic stem cells.

[0444] 3.3 Preparation of scFv format antibody sequences of murine origin

[0445] Without changing the VH, VL sequences, scFv format antibodies 1A6, 1A7, 1A8, 1A9 and 1A10 were constructed using a scheme known in the art, in which the heavy and light chain variable regions were connected by a (G4S)3 linker. The scFv amino acid sequence of scFv format antibody 1A6 is shown as SEQ ID NO: 197. The scFv amino acid sequence of scFv format antibody 1A7 is shown as SEQ ID NO: 198. The scFv amino acid sequence of scFv format antibody 1A8 is shown as SEQ ID NO: 199. The scFv amino acid sequence of scFv format antibody 1A9 is shown as SEQ ID NO: 200. The scFv amino acid sequence of scFv format antibody 1A10 is shown as SEQ ID NO: 201.

[0446] Example 4 Investigation of the internalization of MUC1 β subunit candidate chimeric antibodies

[0447] The internalization of MUC1 β subunit candidate antibodies and positive control antibody H44 was identified by flow cytometry. The tumor cell MUC1 β subunit overexpressing HCT116 cells were counted, resuspended with flow buffer and adjusted to 2 x 10 6 / ml, 30 μl / well were added to a V-bottom 96-well plate, and were divided into experimental and control groups in parallel. 30 μl / well of candidate antibodies and positive control antibodies were added, in which 1A9 and the control antibody were started at a working concentration of 10 μg / ml, diluted with flow buffer at a gradient of 3 times, to form 10 gradients, and the rest of the antibodies and the corresponding control antibodies were started at a working concentration of 30 μg / ml, diluted with flow buffer at a gradient of 4 times, to form 7 gradients, and PBS blank control wells were also set. Incubate at 4°C for 1 hour, wash twice with flow buffer, add anti-human IgG Fc secondary antibody (APC), 30 μl / well, incubate at 4°C for 30 minutes. Wash twice with flow buffer, add 50 μl / well of medium, incubate at 37°C for 1.5 hours. Wash once with flow buffer, add 50 μl / well of quenching buffer (0.2M acetic acid, 0.5M sodium chloride, finally adjust the pH to 2.5) to the experimental group, and place at 4°C for 10 minutes to quench the cell surface fluorescence. The control group was treated in the same way by adding 50 μl / well of flow buffer. Wash twice with flow buffer, shake the cells loose, and add 25 μl / well of flow buffer to the machine. The raw data were plotted and calculated using GraphPad 8.0 software.

[0448] The internalization results are shown in FIG. 7. The MUC1 β subunit antibodies provided herein all have different degrees of endocytosis, indicating that the antibodies described herein have a certain internalization effect and are suitable for preparing ADCs.

[0449] Example 5 Stable expression of MUC1 β subunit specific CAR in T cells

[0450] The CAR structure described in the present application is shown in Figure 8, and the MUC1 β subunit specific CAR structure is composed of anti-human MUC1 β subunit scFV or Fab antibody sequence, human CD8 hinge region (SEQ ID NO: 3), human CD8 transmembrane region (SEQ ID NO: 4), human 4-1BB co-stimulatory domain (SEQ ID NO: 1), human CD3ζ intracellular activation domain (SEQ ID NO: 2), and in addition, a Myc tag (SEQ ID NO: 141) is added at the N-terminus of the CAR structure for easy detection.

[0451] This example investigates the expression of MUC1 specific CAR in human T cells and the stability of CAR-T cell expansion fold and CAR positive rate under routine in vitro culture conditions. The specific method is as follows:

[0452] 1) Thaw the liquid nitrogen frozen human PBMC cells in a 37°C water bath, resuspend and wash three times with 11 ml PBS + 1 ml PBMC system (500g, 5min; 400g, 5min; 300g, 5min), and then mix the washed human PBMC cells with human CD3 magnetic beads (Meiteni, 130-050-101), use a magnetic stand for CD3 positive selection, i.e. separate and retain CD3+ T cells. Resuspend the CD3+ T cells in the culture medium to a cell density of 1E6 cells / ml, add CD3 / CD28 magnetic beads (Summit 40203D) at a ratio of 1:3 (cells: magnetic beads), wash the magnetic beads twice with the culture medium, use a magnetic stand to attract and stand for 1 min, activate the T cells. Add the T cells to the magnetic beads, mix well, add 700 μl of culture medium to each well of a 12-well plate, the cell number is 7E5 cells / well, the density is 1E6 cells / ml, and the sorting day is recorded as Day0.

[0453] 2) After the activated T cells are incubated in a 37°C CO2 incubator for 20 hours, add the corresponding virus supernatant at a virus multiplicity of infection (MOI) of 3, add polybrene to a final concentration of 10 μg / ml, mix well by blowing, and then centrifuge for 1 hour at 1200 rpm using a horizontal centrifuge, place the well plate back in a 37°C CO2 incubator for 24 hours, and the infection day is recorded as Day1.

[0454] 3) After 24h, the cells in each well of the 12-well plate were evenly blown and transferred to a 1.5ml EP tube, centrifuged at 400g for 5min, the supernatant was removed, and the cells were resuspended in 1ml of fresh X-VIVO complete medium to a cell density of 7E5 / ml, and cultured in a 37℃ CO2 incubator. The cells were counted at intervals, and fresh X-VIVO complete medium was added to adjust the cell density to 7E5 / ml. The counting results were statistically analyzed, and the CAR-T cell expansion fold under conventional culture was calculated and plotted using GraphPad 8.0 software.

[0455] 4) CAR-T cell positive rate detection after 6-12 days of culture: the positive rate of the N-terminal Myc tag of the CAR structure was detected by flow cytometry, so as to obtain the CAR expression positive rate.

[0456] The CAR-T expansion results are shown in Figure 9. The CAR-T cells of the present application have a relatively high expansion fold after 9 days of in vitro culture, compared with Mock T cells. The experimental results show that the antibodies provided in the present application have a relatively obvious expansion when prepared into CAR-T cells.

[0457] The CAR expression positive rate results are shown in Figure 10. The CAR-T cells of the present application have a relatively high CAR expression positive rate after in vitro culture. The experimental results show that the antibodies provided in the present application have a stable positive rate performance in the expansion process when prepared into CAR-T.

[0458] Example 6 MUC1 β subunit specific CAR can specifically kill target cells in vitro

[0459] This embodiment evaluates the specific killing ability of CAR-T cells in vitro by constructing a luciferase, MUC1 β subunit overexpressing HCT116 tumor cell line.

[0460] The CAR-T, Mock T and corresponding target cells cultured for 9 days in Example 5 above were centrifuged and resuspended in blank X-VIVO medium, and the target cell density was 1×10 5 / ml, adjust the density of effector cells according to different E:T effector target ratios, then mix and add into sterile V-bottom 96-well plates according to 100 μl target cell suspension + 100 μl CAR-T / Mock T cell suspension per well, set up control wells, and incubate in a 37°C incubator for 24 hours. Centrifuge the 96-well plate at 350g for 5 min, aspirate 100 μl of supernatant, and detect the cytokine using a human IFN-γ ELISA kit (R&D, DY285B). Add 100 μl of luciferase detection substrate (pre-equilibrated to room temperature) to each well of the 96-well plate, and incubate at room temperature for 10 min in the dark. Measure the luciferase content using a multifunctional microplate reader SpectraMax iD3, and calculate the target cell killing percentage according to the formula: target cell killing percentage (%) = (target alone well-experimental well) / target single well x 100%, and process the data using Graphpad prism 8.

[0461] The results of in vitro killing of target cells are shown in Figures 11A and 11B. Relative to the H44 control positive antibody, the CAR-T cells described herein have comparable or slightly better killing percentages. The experimental results show that the antibody provided herein has good killing effect when prepared as CAR-T.

[0462] The results of IFN-γ secretion are shown in Figures 11C and 11D. Relative to Mock T, different degrees of IFN-γ secretion were detected in each group. The experimental results show that the antibody provided herein has the ability to significantly induce IFN-γ secretion when prepared as CAR-T.

[0463] Example 7 ForteBio Octet (biomolecular interaction instrument) verification of competitive binding relationship between candidate chimeric antibody sequences

[0464] To identify whether 1A4, 1A9 antibodies, 1A5, 1A6, 1A8 antibodies can compete with each other for binding. 4 AHC probes (Sartorius, Cat No: 18-5060) were used, each probe as a group, a total of 4 groups. 4 probes were first combined with 100 nM concentration of biotinylated MUC1 β subunit extracellular ECD short peptide, then combined with 100 nM concentration of 1A9, 1A5, 1A6, 1A5 antibodies respectively (Figure 12A-A1 position), and then incubated for 120 s, and then 4 probes were combined with 100 nM+100 nM final concentration of 1A9+1A4, 1A5+1A6, 1A6+1A8, 1A5+1A8 antibody mixture respectively (Figure 12A-A2 position), and then the 4 probes were recycled for 3 times, each time for 5 s, to remove the bound antibodies, and then combined with 100 nM concentration of 1A4, 1A6, 32G11, 32G11 antibodies respectively (Figure 12B-A3 position), and then incubated for 120 s, and then combined with 100 nM+100 nM final concentration of 1A9+1A4, 1A5+1A6, 1A6+1A8, 1A5+1A8 antibody mixture respectively (Figure 12B-A4 position).

[0465] The results of competitive binding are shown in Figures 12A-12B, 1A4, 1A9 antibodies, 1A5, 1A6, 1A8 antibodies can block each other from binding to MUC1 β subunit extracellular ECD short peptide.

[0466] To identify whether 1A4, 1A9 antibodies, 1A5, 1A6, 1A8 antibodies can compete with each other for binding. 4 AHC probes (Sartorius, Cat No: 18-5060) were used, each probe as a group, a total of 4 groups. 4 probes were first combined with 100 nM concentration of biotinylated MUC1 β subunit extracellular ECD short peptide, then combined with 100 nM concentration of 1A9, 1A5, 1A6, 1A5 antibodies respectively (Figure 12A-A1 position), and then incubated for 120 s, and then 4 probes were combined with 100 nM+100 nM final concentration of 1A9+1A4, 1A5+1A6, 1A6+1A8, 1A5+1A8 antibody mixture respectively (Figure 12A-A2 position), and then the 4 probes were recycled for 3 times, each time for 5 s, to remove the bound antibodies, and then combined with 100 nM concentration of 1A4, 1A6, 32G11, 32G11 antibodies respectively (Figure 12B-A3 position), and then incubated for 120 s, and then combined with 100 nM+100 nM final concentration of 1A9+1A4, 1A5+1A6, 1A6+1A8, 1A5+1A8 antibody mixture respectively (Figure 12B-A4 position).

[0467] The results of the competition binding are shown in Figures 12C-12D. The 1A5, 1A6, 1A8 antibodies can prevent the 1A4, 1A9 antibodies from binding to the MUC1 β subunit extracellular ECD short peptide.

[0468] The competition binding relationship between the H44 positive antibody and 1A4, 1A5, 1A9 was identified. Three AHC probes (Sartorius, item number: 18-5060) were used, each probe was a group, and there were three groups. The three probes were first combined with 100 nM concentration of biotinylated MUC1 β subunit extracellular ECD short peptide, and then combined with 100 nM concentration of H44 antibody (Figure 12E-A9 position), and the baseline was briefly balanced for 120 s. Four probes were respectively combined with 100 nM+100 nM final concentration of H44+1A4, H44+1A5, H44+1A9 antibody mixture (Figure 12E-A10 position), and the three probes were recycled for three times, each time for 5 s, to remove the bound antibodies, and then combined with 100 nM concentration of 1A4, 1A5, 1A9 antibody (Figure 12F-A11 position), and the baseline was briefly balanced for 120 s. Three probes were respectively combined with 100 nM+100 nM final concentration of H44+1A4, H44+1A5, H44+1A9 antibody mixture (Figure 12F-A12 position).

[0469] The results of the competition binding are shown in Figures 12E-12F. There is no competition binding relationship between H44 and 1A5 for the MUC1 β subunit extracellular ECD short peptide, and there is a competition binding relationship between H44 and 1A4, 1A9 for the MUC1 β subunit extracellular ECD short peptide.

[0470] In summary, the binding epitopes of the H44 (positive antibody), 1A5, 1A6, 1A8 antibodies are similar epitopes, and the 1A4, 1A9 antibodies are similar epitopes. The antibodies are roughly divided into two epitope groups.

[0471] Example 8 Humanization of the candidate sequence

[0472] The 1A5, 1A6, 1A8, 1A9 and 1A4 sequences were selected, and the human IgG sequences with higher homology were retrieved from the IGMT website (http: / / www.imgt.org / ). The mutations and substitutions of the non-key sites in the framework region were performed to carry out humanization, and the CDR sequences after humanization were not changed.

[0473] The scFv amino acid sequence of the humanized antibody Hu1A5 is shown in SEQ ID NO: 143, the VH amino acid sequence is shown in SEQ ID NO: 151, and the VL amino acid sequence is shown in SEQ ID NO: 167.

[0474] The scFv amino acid sequence of the humanized antibody Hu1A6 is shown as SEQ ID NO: 144, the VH amino acid sequence is shown as SEQ ID NO: 153, and the VL amino acid sequence is shown as SEQ ID NO: 169.

[0475] The scFv amino acid sequence of the humanized antibody Hu1A8 is shown as SEQ ID NO: 145, the VH amino acid sequence is shown as SEQ ID NO: 156, and the VL amino acid sequence is shown as SEQ ID NO: 172.

[0476] The scFv amino acid sequence of the humanized antibody Hu1A9 is shown as SEQ ID NO: 146, the VH amino acid sequence is shown as SEQ ID NO: 158, and the VL amino acid sequence is shown as SEQ ID NO: 174.

[0477] The scFv amino acid sequence of the humanized antibody Hu1A4 is shown as SEQ ID NO: 142, the VH amino acid sequence is shown as SEQ ID NO: 149, and the VL amino acid sequence is shown as SEQ ID NO: 165.

[0478] 8.1 Flow cytometry detection of the binding of the candidate sequences before and after humanization (chimeric full antibody and scFv humanized form) to the MUC1 β subunit on the surface of tumor cells

[0479] HCT116-MUC1 β subunit overexpressing cell counting, resuspend the cells with flow buffer and adjust to 1×10 6 / ml, 30 μl / well of two kinds of cells were added to a V-bottom 96-well plate. 30 μl / well of candidate antibodies before and after humanization and positive control antibody H44 were added, both of which were started at a working concentration of 25 μg / ml, and the flow buffer was diluted at a 2-fold ratio gradient to form 10 gradients, and PBS blank control wells were also set. Incubate at 4°C for 1 hour, wash once with flow buffer, add anti-human IgG Fc secondary antibody (APC), 30 μl / well, incubate at 4°C for 30 minutes. Wash twice with flow buffer, shake the cells loose, add 25 μl / well of flow buffer to the machine. Replace the original data into GraphPad 8.0 software for plotting and calculation.

[0480] The binding activity results to target cells are shown in FIG. 13. The antibodies described in the present application all maintain strong binding activity after humanization, and the binding levels of 1A5, 1A6 and 1A4 antibodies after humanization are maintained at the same level or slightly enhanced as before humanization.

[0481] 8.2 ForteBio Octet (biomolecular interaction instrument) detection of the binding of the candidate sequences before and after humanization (chimeric full antibody and scFv humanized form antibody) to MUC1 β ECD polypeptide

[0482] Dilute the antibody to be verified to 100 nM and add to the 384-well plate. Dilute the biotinylated MUC1 β subunit extracellular ECD short peptide to 100 nM, 2-fold dilution, a total of 7 gradients, and add to the 384-well plate. Use the AHC probe (Sartorius, item number: 18-5060), set the binding time to 300 s, the dissociation time to 360 s, the baseline to 60 s, and the regeneration cycle to 3 times, 5 s each time. Use software to fit the binding-dissociation curve to calculate the affinity of the antibody.

[0483] The affinity results are shown in Table 1. The affinity of the antibodies described in the present application is in the nM level and above, and the affinity does not change much before and after humanization. The experimental results show that the antibodies provided in the present application have strong affinity before and after humanization, and can bind to the MUC1 β subunit extracellular ECD short peptide with high binding activity.

[0484] Table 1. Affinity results

[0485] Example 9. Humanization of 1B2 sequence and binding verification

[0486] In this embodiment, the 1B2 murine monoclonal sequence (SEQ ID NO: 202) against the MUC1 α subunit tumor abnormal glycoform in the invalidated patent CN102264765B is selected as the candidate sequence, and humanization is performed under the premise that the CDR (Kabat code) is not changed, and finally the B21 humanized sequence is obtained. The amino acid sequence of the B21 scFv is shown as SEQ ID NO: 213, the amino acid sequence of the VH is shown as SEQ ID NO: 203, and the amino acid sequence of the VL is shown as SEQ ID NO: 210.

[0487] The change in affinity of B2 before and after humanization is identified using MUC1 overexpressed HCT116 tumor cells. The MUC1 (SEQ ID NO: 217) overexpressed HCT116 tumor cells are counted, resuspended with flow buffer, and adjusted to 1 × 10 6 / ml, 30 μl / well of V-bottom 96-well plates, 30 μl / well of primary antibody (B2, B21-human IgG1 Fc antibody, scFv form), the primary antibody was started with a working concentration of 25 μg / ml, diluted with flow buffer at a 2-fold ratio gradient, forming 11 gradients, and PBS blank control wells were set up, 4-degree incubation for 1 hour, flow buffer cleaning for one time, 30 μl / well of anti-human IgG Fc secondary antibody (APC) was added, 4-degree incubation for 30 minutes. Flow buffer was cleaned for two times, the cells were shaken loose, and 25 μl / well of flow buffer was added for machine. The raw data was substituted into GraphPad 8.0 software for plotting and calculation. Subsequently, CAOV3 ovarian cancer and T47D colon cancer natural cell lines were used to identify the changes in affinity before and after humanization of B2,

[0488] The flow cytometry results are shown in FIGS. 14A-C. FIG. 14A is the affinity result on HCT116 tumor cells, and the experimental results show that the B21 sequence is humanized without changing the CDR amino acid sequence, and the binding force is greatly improved. FIGS. 14B and 14C are the affinity results on CAOV3 ovarian cancer and T47D colon cancer, respectively, and the experimental results show that in the natural cell line, B21 still has very high affinity relative to 1B2. The experimental results show that the MUC1 alpha subunit antibody described in the present application greatly improves the affinity of the antibody while only changing the FR amino acid sequence.

[0489] Example 10 Specific binding of B21 to MUC1

[0490] The specific binding activity of B21 was identified by flow cytometry. HCT116 empty cells and MUC1 overexpressed HCT116 tumor cells were counted, the cells were resuspended with flow buffer and adjusted to 1×10 6 / ml, 30 μl / well of V-bottom 96-well plates, 30 μl / well of primary antibody (B21-mouse Fc antibody, scFv form), the primary antibody was started with a working concentration of 50 μg / ml, diluted with flow buffer at a 2-fold ratio gradient, forming 11 gradients, and PBS negative controls were set up for each antibody, 4-degree incubation for 1 hour, flow buffer cleaning for one time, 30 μl / well of anti-mouse IgG-Fc secondary antibody (AF647) was added, 4-degree incubation for 30 minutes, flow buffer was cleaned for two times, the cells were shaken loose, and 25 μl / well of flow buffer was added for machine. The raw data was substituted into GraphPad 8.0 software for plotting and calculation.

[0491] The flow cytometry results are shown in FIG. 15, and the experimental results show that B21 can specifically bind to MUC1 on the surface of tumor cells, and the MUC1 alpha subunit antibody described in the present application has specificity.

[0492] Example 11 Stable expression of MUC1 a subunit specific B21-CAR (CD8TM) in T cells

[0493] The B21-CAR structure is shown in Figure 16. In addition to the N-terminal CD8 signal peptide SP sequence (SEQ ID NO: 214) and the Flag tag (SEQ ID NO: 215) for easy detection, the MUC1 a subunit specific B21-CAR (CD8TM) in this embodiment is mainly composed of B21 scFv sequence, human CD8 hinge region (SEQ ID NO: 3), human CD8 transmembrane region (SEQ ID NO: 4), human 4-1BB costimulatory domain (SEQ ID NO: 1), and human CD3 zeta intracellular activation domain (SEQ ID NO: 2).

[0494] This example investigates the expression of MUC1 specific B21-CAR in human T cells and the stability of CAR-T cell expansion fold and CAR positive rate under conventional in vitro culture conditions. The culture and test method is shown in Example 5, and the CAR structure N-terminal Flag tag is detected by flow cytometry to obtain the CAR expression positive rate.

[0495] The CAR-T expansion results are shown in Figure 17. The B21-CAR-T cells of the present application have a higher expansion fold after in vitro culture for 9 days compared to Mock T cells. The experimental results show that the MUC1 a subunit antibody provided in the present application has a relatively obvious expansion when prepared into CAR-T cells.

[0496] The CAR expression positive rate results are shown in Figure 18. The B21-CAR-T cells of the present application have a higher CAR expression positive rate after in vitro culture. The experimental results show that the MUC1 a subunit antibody provided in the present application has a stable positive rate performance in the expansion process when prepared into CAR-T.

[0497] Example 12 Activation effect of CA153 positive patient serum on MUC1 a subunit B21-CAR-T

[0498] MUC1 positive tumor patients often have shed MUC1 a subunit (CA153) in their blood, which has been used as a marker for various tumors such as ovarian cancer, breast cancer, etc. in clinical practice. An increase in its level indicates tumor progression and metastasis. This example investigates the activation effect (specifically embodied as prolonging the survival or expansion of the corresponding CAR-T) of free CA153 in the blood on B21-CAR-T.

[0499] X-VIVO medium was used to dilute CA153 ovarian cancer positive patient serum (358 IU / ml, provided by Shanghai First Maternal and Infant Health Hospital) to 1:2, 1:4, 1:8, and B21-CAR-T (CD8TM), MockT, which were routinely cultured for 9 days according to the method of Reference Example 11, were taken 2x10 5

[0500] The CAR-T expansion results are shown in Table 2. It can be seen that CA153 positive serum in the pathological range (the free CA153 level of malignant ovarian cancer patients is mostly 12-250 IU / ml) can not only expand B21-CAR-T (CD8TM) cells in a small amount, but also maintain a high activity of the corresponding CAR-T cells. The experimental results show that the MUC1α subunit antibody described in the application can effectively expand when the CAR-T is prepared in the presence of a certain concentration of CA153 in the environment.

[0501] The activation process of CAR-T involves the recognition and close binding of multiple antigens and CAR structures. By means of the dense physical contact points, i.e. immunological synapses, the activation signal of CAR-T is finally triggered to exert the related functions. The formation of immunological synapses needs a solid surface as a basis to form stable interaction and signal transmission to activate CAR-T. Free antigens are difficult to trigger the activation signal of CAR-T due to the lack of restraint. Considering that CA153 has multiple tandem repeat sequences (VNTR), which support each other, a single CA153 protein can bind to multiple points of VNTR corresponding CAR-T, which may have a certain CAR-T activation effect.

[0502] Table 2 CAR-T expansion results

[0503] Example 13 Specificity of CA153 on B21-CAR-T activation

[0504] The free CA153 in patient serum is generally more than 10 IU / ml. This embodiment further discusses the activation effect of different contents of free CA153 on the corresponding CAR-T, and verifies its specificity.

[0505] X-VIVO medium was used to dilute CA153 serum (150 IU / ml) of an ovarian cancer patient provided by Shanghai First Maternal and Infant Health Hospital to 50 IU / ml and 10 IU / ml, and 2x10 5 ​B21-CAR-T(CD8TM), hu5E6 CAR-T(targeting Claudin18.2 CAR-T, SEQ ID NO: 219) were activated by conventional magnetic beads for 9 days, after centrifugation at 500g for 5 min, 600 μl CA153 diluent was resuspended, placed in a 12-well plate, and cultured in a 37°C incubator for 2 days. After cell counting, the CAR positive rate and the expression of activation markers CD25, CD69 and CD137 were detected by flow cytometry.

[0506] The CAR expression positive rate results are shown in Table 3, and the expression of activation markers is shown in Figures 19A-C. The experimental results show that when CA153 is as low as 10 IU / ml, the CAR expression positive rate is increased, CA153 can still activate B21-CAR-T(CD8TM) to a certain extent, and is positively correlated with the concentration of CA153, while Hu5E6-CAR-T has no obvious activation effect. The experimental results show that the MUC1α subunit antibody described in the application has a good CAR expression positive rate when it is prepared as CAR-T in the presence of a certain concentration of CA153 in the environment, and can be activated by CA153 in the environment, and shows a concentration-dependent effect.

[0507] Table 3. CAR expression positive rate results

[0508] Example 14 Confirmation of the expression of MUC1α and β subunits in ovarian cancer tissues of stages III-IV

[0509] In order to further confirm the target abundance in advanced patients, B21-mouse Fc(targeting α subunit abnormal glycosylation antigen) and Hu1A5-rabbit Fc antibody(targeting β subunit produced after cleavage) were used as primary antibodies for immunohistochemical colocalization fluorescence experiments on ovarian cancer tissue chip F100Ov02(48 cases of serous carcinoma and 2 cases of ovarian tissue, Zhongke Guanghua) to confirm the shedding of MUC1α subunit in advanced ovarian cancer tissues. After incubation with the primary antibody, the corresponding goat anti-mouse Fc(FITC, green light) and rabbit Fc(CY3, red light) fluorescent antibodies were used for secondary antibody staining, and B21 and Hu1A5 colocalization emitted yellow light.

[0510] The cell membrane staining intensity and positive rate results are shown in FIGS. 20A-B. The obvious horizontal band in the figure represents a sample, and the numbers in the figure are the staining intensity score and positive rate (%) results of the specific sample. The horizontal coordinate represents different antibody staining, in which B21+Hu1A5 represents double positive staining of the two antibodies, B21 / Hu1A5 represents positive staining of B21 or Hu1A5, Hu1A5-B21 represents Hu1A5 single staining, and the vertical coordinate represents the tumor stage of different samples. As shown in FIG. 20A, the staining intensity of B21, Hu1A5 antibody and B21, Hu1A5 co-staining does not differ much overall. As shown in FIG. 20B, compared with Hu1A5, B21 shows a larger proportion of positive, indicating that there are still a large number of un-shed MUC1 a subunits in advanced ovarian cancer tissues. The relative positive rate of Hu1A5 shows an increasing relationship between III and IV patients, indicating that a larger proportion of a subunits are shed in IV patients. Referring to the Hu1A5 single staining results, there are tumor cell types in which the a subunit is completely shed in some samples of advanced ovarian cancer. Considering the coexistence of a subunit and β subunit in advanced ovarian cancer, targeting the MUC1 a subunit and β subunit can cover more tumor types.

[0511] Example 15 Difference in activation of MUC1 single epitope and double epitope CAR-J after receiving stimulation of target cells with different MUC1 forms

[0512] For B21 (targeting a subunit abnormal glycosylation antigen) and Hu1A5 antibody (targeting β subunit produced after cleavage), MUC1 single epitope and double epitope CAR structures were constructed in this embodiment, and Jurkat-NFAT-GFP cells were transduced to construct CAR-J effector cells. The structure schematic diagram is shown in FIG. 21. In addition, wild-type CAOV3, SKOV3 and overexpression cell lines SKOV3-MUC1β, SKOV3-MUC1β-MUC1 full-length protein were used as target cells to simulate the conditions of a not shedding, partial shedding and basically complete shedding in the group. The target abundance was detected by flow cytometry, and the results are shown in FIGS. 22A-B.

[0513] When CAR-J cells receive stimulation of target cells with different MUC1 forms, downstream promoters NFAT can activate GFP expression to different degrees. The schematic diagram is shown in FIG. 23, in which the GFP expression intensity is proportional to the degree of CAR activation, and the rationality of the CAR structure can be evaluated accordingly. The specific operation is as follows: according to the effector target ratio of 1:3, 0.6E5 effector cells and 1.8E5 target cells were taken, 1.2 ml of 1640 culture medium containing 10% FBS was resuspended into a 24-well plate, and an effector cell control well was set. After 24 h of incubation in a 37°C CO2 incubator, the GFP MFI was detected by flow cytometry. The experiment wells with and without target cells were compared and plotted.

[0514] The activation degree results are shown in Figure 24. The experimental results show that under the same target cell stimulation, the dual epitope CAR-J has higher signal activation intensity than the single epitope CAR-J, which implies that the CAR structure has higher potential. The experimental results show that the MUC1 a subunit antibody and the MUC1 β subunit antibody described in the application can be better activated after being prepared into a dual epitope CAR-T.

[0515] Example 16 In vitro killing activity and cytokine secretion during killing of dual epitope CAR-T targeting MUC1 a, β subunits (two Donor T cells)

[0516] The specific killing ability of dual epitope CAR-T cells targeting MUC1 a subunit (B21) and β subunit (Hu1A5) in vitro was evaluated by wild type SKOV3 and overexpression cell lines SKOV3-MUC1 β and SKOV3-MUC1 β-MUC1 full-length protein.

[0517] 16.1 24h in vitro killing and cytokine secretion

[0518] The culture method is shown in Example 5. The CAR-T, MockT and corresponding target cells cultured for 7 days were centrifuged and resuspended with blank X-VIVO medium. The target cell density was 1x10 5 / ml. The effector cell density was adjusted according to different E:T ratios, and then 100 μl of target cell suspension + 100 μl of CAR-T / MockT cell suspension was added to a sterile V-bottom 96-well plate. Control wells were set up, and the plate was incubated in a 37°C incubator for 24 hours. The 96-well plate was centrifuged at 350g for 5 min, and 100 μl of supernatant was removed. The human IFN-γ ELISA kit (R&D, DY285B) was used to detect the cytokine secretion level of the E:T ratio 1:1 experimental group.

[0519] 100 μl of luciferase detection substrate (pre-equilibrated to room temperature) was added to each well of the 96-well plate, and the plate was incubated at room temperature for 10 min. The multifunctional enzyme label instrument SpectraMax iD3 was used to detect the content of luciferase. At the same time, the formula: target cell killing percentage (%) = (target cell alone well - CAR-T / target cell mixed experimental well) / target cell alone well x 100 was used to calculate the target cell killing percentage. The data was processed by Graphpad prism 8.

[0520] The cytokine secretion results are shown in Figures 25-26. The double epitope CAR-T group has higher IL-2 and IFN-γ secretion relative to the single epitope CAR-T. The experimental results show that the MUC1 α subunit antibody and MUC1 β subunit antibody described in the application have stronger ability to induce cytokine secretion after being prepared into double epitope CAR-T.

[0521] The target cell killing results in vitro are shown in Figure 27. The double epitope CAR-T group has higher percentage of killing of the same target cells relative to the single epitope CAR-T. The experimental results show that the MUC1 α subunit antibody and MUC1 β subunit antibody described in the application have stronger target cell killing ability after being prepared into double epitope CAR-T.

[0522] 16.2 Continuous multiple rounds of cytokine secretion

[0523] The culture method is shown in Example 5. The CAR-T, MockT and corresponding target cells are routinely cultured for 9 days, then centrifuged and resuspended with blank X-VIVO medium, and the E:T is adjusted to 1:3 (target cells 2E5), then 600 μl of target cell suspension + 600 μl of CAR-T / MockT cell suspension is added to a sterile 12-well plate, and cultured in a 37°C incubator for 4 days. After 4 days, 100 μl of supernatant is aspirated, the cytokine secretion level is detected by human IFN-γ ELISA kit (R&D, DY285B), and the required amount of effector cells is taken out and plated as described above, and this is repeated for 3 rounds.

[0524] The multiple rounds of target cell killing results in vitro are shown in Figure 27. The double epitope CAR-T has significantly higher cytokine secretion than the single epitope CAR-T on different abundance of target cell types, and the 24h and continuous multiple rounds of cytokine secretion are also this trend. The experimental results show that the MUC1 α subunit antibody and MUC1 β subunit antibody described in the application have stronger ability to induce cytokine secretion when continuously killing target cells after being prepared into double epitope CAR-T, and have better drug potential.

[0525] Example 17 CA153 positive patient serum, ascites specific activation of MUC1 double epitope B21+Hu1A5-CAR-T

[0526] CA153 ovarian cancer positive patient serum (from two patients) and ascites (from one patient) (provided by Shanghai First Maternal and Infant Health Hospital) were diluted to a final concentration of 100 IU / ml of CA153 using X-VIVO medium, CA153 protein (CD BioGlycom, XCA-027Q) was diluted to a final concentration of 200 IU / ml and 100 IU / ml, and a B21 antibody experimental group was set up, as shown in Table 4. The culture method was as shown in Example 5, and B21 + Hu1A5-CAR-T was routinely cultured for 8 days. 2 x 10 5

[0527] CAR expression positive rate results are shown in Figures 29A-C and 30A-C, and the expression of activation markers is shown in Figures 29D-F and 30D-F. The experimental results show that in the pathological concentration range, the MUC1 double epitope CAR has a good positive expression rate, and CA153 can specifically activate the MUC1 double epitope CAR-T. The experimental results show that the MUC1 alpha subunit antibody and the MUC1 beta subunit antibody described in the application have a good CAR expression positive rate when prepared into a double epitope CAR-T under different pathological conditions, and can be activated by CA153 in the environment.

[0528] Table 4 Grouping

[0529] Example 18 In vivo efficacy of double epitope CAR-T and CA153

[0530] 18.1 Confirmation of target cell abundance in vivo

[0531] CAOV3 natural ovarian cancer cells were inoculated at a concentration of 3 x 10 6 2

[0532] The immunohistochemical staining results show that the single staining and double staining intensities of B21 and Hu1A5 antibodies in tumor cells are 3, the single positive proportion of B21 is 80%, the single positive proportion of Hu1A5 is 55%, and the double positive proportion is 30%. This is different from the CAOV3 cell line cultured in vitro, and may be related to the tumor microenvironment. Overall, the CAOV3 tumor-bearing mouse target is more consistent with the clinical patient situation in Example 14, and can be used for subsequent animal experiment evaluation. ​​​

[0533] 18.2 Evaluation of in vivo efficacy

[0534] CAOV3 ovarian cancer cells were inoculated subcutaneously on the right side of B-NDG mice at a concentration of 3 x 10 6 After the tumors grew to about 280 mm 2 , the mice were randomly divided into 4 groups. Mock T, B21-CAR-T, Hu1A5-CAR, and B21-Hu1A5-CAR-T cells were injected intravenously at a dose of 1E7 per mouse. The CA153 administration group was injected intravenously with 360 IU of CA153 protein per mouse (mouse blood volume was calculated as 1.8 ml, and serum CA153 concentration was estimated as 200 IU / ml). During the experiment, if any one or more of the following conditions occurred, the experimental animals were euthanized: 1) the tumor was severely damaged and did not scab within 3 days; 2) the animal exhibited abnormal movement or paralysis; 3) the animal's tumor reached 2000 mm 3 . During the administration and observation period, the mice's body weight and tumor volume were measured 3 times per week, and the measurement values were recorded. At the end of the experiment, the animals were euthanized.

[0535] The results of the in vivo efficacy evaluation are shown in FIG. 31. In this animal model, compared with single epitope CAR-T, the dual epitope B21-Hu1A5-CAR-T had a more obvious tumor inhibition effect. The experimental results showed that the MUC1 a subunit antibody and the MUC1 b subunit antibody described in the present application had good in vivo target cell killing ability when prepared as a dual epitope CAR-T, and could better inhibit tumor growth.

Claims

1. An isolated antigen binding protein capable of binding to a MUC1 beta subunit, wherein the antigen binding protein comprises an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising a heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 186, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 187, or SEQ ID NO: 188, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 189, or SEQ ID NO:

190.

2. The antigen binding protein of claim 1, wherein the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-12, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 15-21, and the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24-31.

3. The antigen binding protein of any one of claims 1-2, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations: (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 15, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 24; (2) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 16, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 25; (3) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 17, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 26; (4) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 27; (5) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 19, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 28; (6) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 29; (7) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 20, the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 30; and (8) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 21, the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO:

31.

4. The antigen binding protein of any one of claims 1-3, wherein the VH comprises a H-FR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 34-44, a H-FR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 47-55, a H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 58-70, and a H-FR4 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 72-76.

5. The antigen binding protein of any one of claims 1-4, wherein the VH comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4 selected from any one of the following combinations: (1) the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 34, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 47, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 58, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 73; (2) the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 34, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 48, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 59, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 72; (3) the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 35, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 48, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 60, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 74; (4) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 47, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 61, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 73; (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 62, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74; (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 38, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 50, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 63, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 75; (7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 39, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 51, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 64, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74; (8) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 52, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 65, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 76; (9) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 52, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 66, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 76; (10) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 51, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74; (11) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 68, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 75; (12) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 43, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 54, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 69, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 74; and (13) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 44, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 70, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:

75.

6. The antigen binding protein of any one of claims 1-5, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 147-159.

7. The antigen binding protein of any one of claims 1-6, wherein the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77-80, SEQ ID NO: 83, or SEQ ID NO: 191, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 192, and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 90-92, SEQ ID NO: 94, or SEQ ID NO:

193.

8. The antigen binding protein of claim 7, wherein the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77-83, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 84-89, and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 90-96.

9. The antigen binding protein of any one of claims 7-8, wherein the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are selected from any one of the following combinations: (1) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 84, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 90; (2) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 91; (3) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 92; (4) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 93; (5) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94; (6) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 89, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95; or (7) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

91. (3) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 79, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 86, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 92; (4) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 93; (5) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 81, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 94; (6) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 94; (7) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 83, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 89, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 95; and (8) the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO:

96.

10. The antigen binding protein of any one of claims 7-9, wherein the VL comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 97-108, the L-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 109-118, the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 119-130, and the L-FR4 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 131-139.

11. The antigen binding protein of any one of claims 7-10, wherein the VL comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1, L-FR2, L-FR3, and L-FR4 are selected from any one of the following combinations: (1) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 97, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 109, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 119, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 131; (2) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 98, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 110, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 120, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 131; (3) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 99, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 111, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 121, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 132; (4) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 100, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 112, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 122, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 131; (5) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 101, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 112, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 123, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 132; (6) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 102, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 113, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 124, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 133; (7) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 103, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 114, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 125, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 134; (8) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 104, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 115, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 126, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 135; (9) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 105, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 115, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 126, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 135; (10) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 106, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 116, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 127, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 136; (11) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 107, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 117, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 128, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 137; (12) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 108, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 118, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 129, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 138; and (13) the L-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 102, the L-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 113, the L-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 130, and the L-FR4 comprises an amino acid sequence set forth in SEQ ID NO:

139.

12. The antigen binding protein of any one of claims 7-11, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 163-175.

13. The antigen binding protein of any one of claims 1-12, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of the HCDR1, HCDR2 and HCDR3, and the LCDR1, LCDR2 and LCDR3 are selected from any one of the following combinations: (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 77, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 84, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 90; (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 78, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 91; (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 86, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 92; (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 93; (5) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94; (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 88, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 94; (7) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 89, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95; and (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 87, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

96.

14. The antigen binding protein of any one of claims 1-13, wherein the amino acid sequences of the VH and VL are selected from any one of the following combinations: (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 147, the VL comprises the amino acid sequence set forth in SEQ ID NO: 163; (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 148, the VL comprises the amino acid sequence set forth in SEQ ID NO: 164; (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 149, the VL comprises the amino acid sequence set forth in SEQ ID NO: 165; (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 150, the VL comprises the amino acid sequence set forth in SEQ ID NO: 166; (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 151, the VL comprises the amino acid sequence set forth in SEQ ID NO: 167; (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 152, the VL comprises the amino acid sequence set forth in SEQ ID NO: 168; (7) the VH comprises an amino acid sequence set forth in SEQ ID NO: 153, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 169; (8) the VH comprises an amino acid sequence set forth in SEQ ID NO: 154, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 170; (9) the VH comprises an amino acid sequence set forth in SEQ ID NO: 155, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 171; (10) the VH comprises an amino acid sequence set forth in SEQ ID NO: 156, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 172; (11) the VH comprises an amino acid sequence set forth in SEQ ID NO: 157, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 173; (12) the VH comprises an amino acid sequence set forth in SEQ ID NO: 158, and the VL comprises an amino acid sequence set forth in SEQ ID NO: 174; and (13) the VH comprises an amino acid sequence set forth in SEQ ID NO: 159, and the VL comprises an amino acid sequence set forth in SEQ ID NO:

175.

15. The antigen binding protein of any one of claims 1-14, wherein the antigen binding protein further comprises an immunoglobulin constant region.

16. The antigen binding protein of claim 15, wherein the immunoglobulin constant region is a heavy chain constant region of human IgG and / or a light chain constant region of human antibody.

17. The antigen binding protein of claim 16, wherein the heavy chain constant region of human IgG is a heavy chain constant region of human IgGl, IgG2, IgG3, or IgG4.

18. The antigen binding protein of claim 16, wherein the light chain constant region of human antibody is a human Kappa (K) or Lambda (L) light chain constant region.

19. The antigen binding protein of any one of claims 1-18, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

20. The antigen binding protein of claim 19, wherein the antigen binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

21. The antigen binding protein of any one of claims 1-20, which is a scFv comprising an amino acid sequence set forth in any one of SEQ ID NOs: 142-146 or SEQ ID NOs: 194-201.

22. An isolated antigen binding protein that is capable of binding to a MUC1 beta subunit, wherein the antigen binding protein comprises a VH comprising a heavy chain complementarity determining region HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 5, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 13, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO:

22.

23. The antigen binding protein of claim 22, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations: (1) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 6, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 14, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 23; and (2) the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 5, the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 13, and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO:

22.

24. The antigen binding protein of any one of claims 22-23, wherein the VH comprises a H-FR1 comprising an amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 32, a H-FR2 comprising an amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 45, a H-FR3 comprising an amino acid sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 56, and a H-FR4 comprising an amino acid sequence set forth in SEQ ID NO: 72 or SEQ ID NO:

71.

25. The antigen binding protein of any one of claims 22-24, wherein the VH comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4 selected from any one of the following combinations: (1) the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 33, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 46, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 57, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO: 72; and (2) the H-FR1 comprises an amino acid sequence set forth in SEQ ID NO: 32, the H-FR2 comprises an amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 comprises an amino acid sequence set forth in SEQ ID NO: 56, and the H-FR4 comprises an amino acid sequence set forth in SEQ ID NO:

71. (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 45, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 56, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:

71.

26. The antigen binding protein of any one of claims 22-25, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO:

160.

27. The antigen binding protein of any one of claims 22-26, wherein the antigen binding protein further comprises an immunoglobulin constant region.

28. The antigen binding protein of claim 27, wherein the immunoglobulin constant region is a human immunoglobulin Fc region.

29. The antigen binding protein of any one of claims 22-28, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

30. The antigen binding protein of claim 29, wherein the antigen binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

31. The antigen binding protein of any one of claims 22-30, which is a VHH comprising the amino acid sequence set forth in SEQ ID NO: 161 or SEQ ID NO:

160.

32. An isolated antigen binding protein capable of binding to a MUC1 alpha subunit, wherein the antigen binding protein comprises an antibody heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence set forth in SEQ ID NO:

203.

33. The antigen binding protein of claim 32, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:

210.

34. The antigen binding protein of any one of claims 32-33, wherein the antigen binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 203 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

210.

35. The antigen binding protein of any one of claims 32-34, wherein the antigen binding protein further comprises an immunoglobulin constant region.

36. The antigen binding protein of claim 35, wherein the immunoglobulin constant region is a heavy chain constant region of a human IgG and / or a light chain constant region of a human antibody.

37. The antigen binding protein of claim 36, wherein the heavy chain constant region of a human IgG is a heavy chain constant region of a human IgGl, IgG2, IgG3, or IgG4.

38. The antigen binding protein of claim 36, wherein the light chain constant region of a human antibody is a human kappa (Kappa) or lambda (Lambda) light chain constant region.

39. The antigen binding protein of any one of claims 32-38, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

40. The antibody or antigen binding fragment thereof of claim 39, wherein the antigen binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

41. The antigen binding protein of any one of claims 32-40, which is a scFv comprising the amino acid sequence set forth in SEQ ID NO:

213.

42. A chimeric antigen receptor comprising the antigen binding protein of any one of claims 1-41.

43. The chimeric antigen receptor of claim 42, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

44. The chimeric antigen receptor of claim 43, wherein the antigen binding domain comprises the antigen binding protein of any one of claims 1-41.

45. The chimeric antigen receptor of any one of claims 43-44, wherein the transmembrane domain comprises a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

46. The chimeric antigen receptor of any one of claims 43-45, wherein the transmembrane domain is derived from a transmembrane domain of CD8.

47. The chimeric antigen receptor of any one of claims 43-46, wherein the costimulatory domain comprises a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

48. The chimeric antigen receptor of any one of claims 43-47, wherein the costimulatory domain is derived from a 4-1BB costimulatory domain.

49. The chimeric antigen receptor of any one of claims 43-48, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from any one of the following proteins: CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon Rl gamma, Fc epsilon Rl beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

50. The chimeric antigen receptor of any one of claims 43-49, wherein the intracellular signaling domain is derived from an intracellular signaling domain of CD3 zeta.

51. The chimeric antigen receptor of any one of claims 43-50, wherein the chimeric antigen receptor further comprises a hinge region.

52. The chimeric antigen receptor of claim 51, wherein the hinge region comprises a hinge region derived from any one of the following proteins: CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon Rl gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

53. The chimeric antigen receptor of any one of claims 51-52, wherein the hinge region is derived from a hinge region of CD8.

54. The chimeric antigen receptor of any one of claims 42-53, comprising a CD8 alpha signal peptide, an antigen binding protein targeting the MUC1 alpha subunit or beta subunit, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

55. A modified immune cell comprising the chimeric antigen receptor of any one of claims 42-54.

56. The modified immune cell of claim 55, wherein the immune cell is a T cell, an NK cell, an iNKT cell, a dendritic cell, and / or a macrophage.

57. An immunoconjugate, wherein the immunoconjugate comprises the antigen binding protein of any one of claims 1-41.

58. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1-41 and / or the chimeric antigen receptor of any one of claims 42-54.

59. A vector comprising the nucleic acid molecule of claim 58.

60. A cell comprising the nucleic acid molecule of claim 58 and / or the vector of claim 59.

61. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, and / or the cell of claim 60, and optionally a pharmaceutically acceptable carrier.

62. A detection kit comprising the antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, the cell of claim 60, and / or the pharmaceutical composition of claim 61, for use in detecting the presence and / or amount of MUC1 β subunit or MUC1 α subunit in a sample or subject.

63. A method of detecting the presence and / or amount of MUC1 β subunit or MUC1 α subunit, comprising using the antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, the cell of claim 60, and / or the pharmaceutical composition of claim 61.

64. Use of the antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, the cell of claim 60, and / or the pharmaceutical composition of claim 61, for the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

65. The use according to claim 64, wherein the disease and / or disorder is a tumor.

66. The use according to claim 65, wherein the tumor is a solid tumor and / or a hematological tumor.

67. The use according to any one of claims 65-66, wherein the tumor is positive for MUC1 β subunit and / or MUC1 α subunit expression.

68. The use according to any one of claims 65-67, wherein the tumor is an epithelial-derived MUC1 β subunit and / or MUC1 α subunit positive tumor.

69. The use according to any one of claims 65-68, wherein the tumor is a breast cancer, a prostate cancer, a colorectal cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, a gastric cancer, a myeloma, a liver cancer, and / or a biliary duct cancer.

70. A method of preventing and / or treating a disease and / or disorder comprising administering to a subject in need thereof the antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, the cell of claim 60, and / or the pharmaceutical composition of claim 61.

71. The method of claim 70, wherein the disease and / or disorder is a tumor.

72. The method of claim 71, wherein the tumor is a solid tumor and / or a hematological tumor.

73. The method of any one of claims 71-72, wherein the tumor is positive for MUC1 β subunit and / or MUC1 α subunit expression.

74. The method of any one of claims 71-73, wherein the tumor is an epithelial-derived MUC1 β subunit and / or MUC1 α subunit positive tumor.

75. The method of any one of claims 71-74, wherein the tumor is a breast cancer, a prostate cancer, a colorectal cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, a gastric cancer, a myeloma, a liver cancer, and / or a biliary cancer.

76. The antigen binding protein of any one of claims 1-41, the chimeric antigen receptor of any one of claims 42-54, the modified immune cell of any one of claims 55-56, the immunoconjugate of claim 57, the nucleic acid molecule of claim 58, the vector of claim 59, the cell of claim 60, and / or the pharmaceutical composition of claim 61 for use in the prevention and / or treatment of a disease and / or disorder.

77. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of claim 76, wherein the disease and / or disorder is a tumor.

78. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of claim 77, wherein the tumor is a solid tumor and / or a hematological tumor.

79. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of any one of claims 77-78, wherein the tumor is positive for MUC1 β subunit and / or MUC1 α subunit expression.

80. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of any one of claims 77-79, wherein the tumor is an epithelial-derived MUC1 β subunit and / or MUC1 α subunit positive tumor.

81. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of any one of claims 77-80, wherein the tumor is a breast cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, gastric cancer, myeloma, liver cancer, and / or biliary duct cancer.

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