Anti-AFP / HLA02 TCR-like antibodies and uses thereof

Abstract

The present application relates to anti-AFP / HLA02 TCR-like antibodies and uses thereof, wherein the antibodies have a K of about 3.1E-09M or less. D Human AFP values 158-166 The present application further relates to a chimeric antigen receptor comprising said antibody, an immune complex, and a cell comprising said chimeric antigen receptor.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals, and in particular to anti-AFP / HLA02 TCR-like antibodies and uses thereof. [Background technology]

[0002] Alpha-fetoprotein (AFP) is a glycoprotein. In adults, serum AFP levels are elevated in approximately 80% of liver cancer patients, the AFP positivity rate is 50% in germ cell tumors, and AFP levels are elevated to varying degrees in patients with pancreatic or lung cancer and liver cirrhosis. Therefore, AFP is expected to be a target for treating various solid tumors.

[0003] Intracellular tumor-specific antigens are processed into peptides and presented on the surface of tumor cells via major histocompatibility complex (MHC) class I. TCR-like antibodies bind to the polypeptide / MHC complexes, inducing tumor cell death. The TCR-like antibodies are converted into CAR structures, allowing T cells to mediate specific tumor lysis.

[0004] While CART technology has achieved great success in hematological malignancies, no significant changes have been seen in its application in solid tumors. Challenges with CART therapy include: 1) unlike leukemia, solid tumors are not ubiquitous throughout the body; CART cells must reach the tumor site and infiltrate the tumor before they can be effective. This means that the tissue structure of solid tumors creates spatial barriers to CART therapy; 2) the immune microenvironment within the tumor inhibits CART cells from functioning normally, leading to T cell depletion; and 3) conditions within the solid tumor microenvironment, such as hypoxia and lack of nutrients, are unfavorable for the proliferation of CART cells and the generation of targeted cytotoxicity.

[0005] Therefore, there is a pressing need for the development of novel antibodies and CAR constructs to treat solid tumors. Summary of the Invention

[0006] The present application provides isolated antigen-binding proteins, which have the following properties: 1) a K of about 3.1E-09 M or less; D Human AFP values 158-166 / HLA-A02*01* complex, and 2) mouse AFP 158 / / Ability to bind to the HLA-A02*01* complex.

[0007] In one aspect, the present application provides an isolated antigen binding protein, wherein said antigen binding protein comprises an HCDR3, wherein said HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56.

[0008] In some embodiments, the antigen binding protein comprises an HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO:55.

[0009] In some embodiments, the antigen binding protein comprises an HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:50.

[0010] In some embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3 of a heavy chain variable region set forth in any one of SEQ ID NOs:72 to 84.

[0011] In some embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:56; the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO:55; and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:50.

[0012] In some embodiments, the HCDR1, HCDR2, and HCDR3 are: a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19, b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35; e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30, h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46, i) HCDR1: SEQ ID NO: 50, HCDR2: SEQ ID NO: 51, and HCDR3: SEQ ID NO: 52; j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56; and k) HCDR1: comprising any one group of amino acid sequences selected from SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30.

[0013] In some embodiments, the antigen-binding protein comprises H-FR1, wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65.

[0014] In some embodiments, the antigen binding protein comprises an H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:66.

[0015] In some embodiments, the antigen binding protein comprises an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69.

[0016] In some embodiments, the antigen binding protein comprises an H-FR4, wherein the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

[0017] In some embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs:58 to 65, H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:66, H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs:67 to 69, and H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs:70 to 71.

[0018] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the antigen binding protein are a) H-FR1: SEQ ID NO: 58, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, b) H-FR1: SEQ ID NO: 59, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, c) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, d) H-FR1: SEQ ID NO: 61, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; e) H-FR1: SEQ ID NO: 62, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, f) H-FR1: SEQ ID NO: 63, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, g) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, h) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 68, and H-FR4: SEQ ID NO: 71, and i) Contains any one group of amino acid sequences selected from H-FR1: SEQ ID NO: 65, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 69, and H-FR4: SEQ ID NO: 70.

[0019] In some embodiments, the antigen-binding protein comprises a heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NO:72 to SEQ ID NO:84.

[0020] In some embodiments, the antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0021] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb.

[0022] In some embodiments, the antigen binding protein comprises a VHH or an antigen binding fragment thereof.

[0023] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, and a fully human antibody.

[0024] In some embodiments, the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:72 to SEQ ID NO:84.

[0025] In another aspect, the present application provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a targeting moiety, wherein the targeting moiety comprises an HCDR3, and wherein the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56.

[0026] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55.

[0027] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50.

[0028] In some embodiments, the targeting portion of the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOs: 72 to 84.

[0029] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:56; the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO:55; and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:50.

[0030] In some embodiments, the HCDR1, HCDR2, and HCDR3 in the chimeric antigen receptor are a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19, b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35; e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30, h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46, i) HCDR1: SEQ ID NO: 50, HCDR2: SEQ ID NO: 51, and HCDR3: SEQ ID NO: 52; j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56; and k) HCDR1: comprising any one group of amino acid sequences selected from SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30.

[0031] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR1, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65.

[0032] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 66.

[0033] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69.

[0034] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR4, the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

[0035] In some embodiments, the targeting portion of the chimeric antigen receptor comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65, H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69, and H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

[0036] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the chimeric antigen receptor are a) H-FR1: SEQ ID NO: 58, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, b) H-FR1: SEQ ID NO: 59, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, c) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, d) H-FR1: SEQ ID NO: 61, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; e) H-FR1: SEQ ID NO: 62, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, f) H-FR1: SEQ ID NO: 63, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, g) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, h) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 68, and H-FR4: SEQ ID NO: 71, and i) Contains any one group of amino acid sequences selected from H-FR1: SEQ ID NO: 65, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 69, and H-FR4: SEQ ID NO: 70.

[0037] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises an antibody or antigen-binding fragment.

[0038] In some embodiments, the antigen-binding fragment in the chimeric antigen receptor is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0039] In some embodiments, the targeting moiety in the chimeric antigen receptor comprises a VHH.

[0040] In some embodiments, the VHH in the chimeric antigen receptor is a human AFP. 158-166 / Targeting the HLA-A02*01* complex.

[0041] In some embodiments, the targeting portion of the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 72 to 84.

[0042] In some embodiments, the chimeric antigen receptor comprises a hinge region.

[0043] In some embodiments, the hinge region in the chimeric antigen receptor comprises a hinge region derived from the following proteins: IgG4, IgG1, and CD8.

[0044] In some embodiments, the hinge region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 147.

[0045] In some embodiments, the chimeric antigen receptor comprises a transmembrane domain.

[0046] In some embodiments, the transmembrane domain in the chimeric antigen receptor comprises a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154, and SLAM, or a combination thereof.

[0047] In some embodiments, the transmembrane domain in the chimeric antigen receptor comprises a transmembrane domain derived from CD8.

[0048] In some embodiments, the transmembrane domain in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 149.

[0049] In some embodiments, the N-terminus of the transmembrane domain in the chimeric antigen receptor is linked to the C-terminus of the hinge region.

[0050] In some embodiments, the chimeric antigen receptor comprises a costimulatory signaling domain.

[0051] In some embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain from a protein 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, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88, or a combination thereof.

[0052] In some embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from 4-1BB.

[0053] In some embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from CD28.

[0054] In some embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 132 or SEQ ID NO: 130.

[0055] In some embodiments, the N-terminus of the costimulatory signaling domain in the chimeric antigen receptor is linked to the C-terminus of the transmembrane domain.

[0056] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling domain.

[0057] In some embodiments, the intracellular signaling domain in the chimeric antigen receptor comprises an intracellular signaling domain derived from a protein selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, and DAP-12, or a combination thereof.

[0058] In some embodiments, the intracellular signaling domain in the chimeric antigen receptor comprises an intracellular signaling domain derived from CD3ζ.

[0059] In some embodiments, the intracellular signaling domain in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 134.

[0060] In some embodiments, the N-terminus of the intracellular signaling domain in the chimeric antigen receptor is linked to the C-terminus of the costimulatory signaling domain.

[0061] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 112 to 124.

[0062] In another aspect, the present application provides a polypeptide comprising the isolated antigen binding protein and / or the chimeric antigen receptor.

[0063] In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein.

[0064] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.

[0065] In another aspect, the present application provides one or more vectors comprising the isolated nucleic acid molecule described above.

[0066] In another aspect, the present application provides one or more modified cells comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, and / or the vector.

[0067] In some embodiments, the modification comprises upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the cell.

[0068] In some embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

[0069] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof is from a human.

[0070] In some embodiments, the functional fragment comprises a fragment or truncation of the low density lipoprotein receptor-related protein that has the low density lipoprotein receptor-related protein activity.

[0071] In some embodiments, the low density lipoprotein receptor-related protein comprises low density lipoprotein receptor-related protein 6 and truncations thereof, and / or low density lipoprotein receptor-related protein 5 and truncations thereof.

[0072] In some embodiments, the truncated form of low-density lipoprotein receptor-related protein 6 comprises the intracellular region of low-density lipoprotein receptor-related protein 6, and / or the truncated form of low-density lipoprotein receptor-related protein 5 comprises the intracellular region of low-density lipoprotein receptor-related protein 5.

[0073] In some embodiments, the truncated form of low-density lipoprotein receptor-related protein 6 comprises the transmembrane region of low-density lipoprotein receptor-related protein 6 and the LDLR region of low-density lipoprotein receptor-related protein 6, and / or the truncated form of low-density lipoprotein receptor-related protein 5 comprises the transmembrane region of low-density lipoprotein receptor-related protein 5 and the LDLR region of low-density lipoprotein receptor-related protein 5.

[0074] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, and SEQ ID NO:144.

[0075] In some embodiments, the modification comprises the introduction into the modified cell of a vector that leads to up-regulation of expression levels of the low density lipoprotein receptor-related protein or fragment thereof.

[0076] In some embodiments, the modified cells comprise immune cells.

[0077] In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0078] In some embodiments, the modified cells comprise T cells.

[0079] In another aspect, the present application provides a method for producing the isolated antigen binding protein, the chimeric antigen receptor, and / or the polypeptide, the method comprising culturing the modified cell under conditions allowing expression of the isolated antigen binding protein and / or the polypeptide.

[0080] In another aspect, the present application provides one or more pharmaceutical compositions comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the modified cell, and / or a pharmaceutically acceptable adjuvant and / or excipient.

[0081] In another aspect, the present application provides: A method for detecting an AFP protein is provided, comprising administering the isolated antigen-binding protein, the polypeptide, or the immune complex.

[0082] In another aspect, the present application provides a kit for detecting an AFP protein, comprising the isolated antigen-binding protein, the polypeptide, or the immune complex.

[0083] In another aspect, the present application provides the use of the isolated antigen-binding protein, the polypeptide, or the immune complex in the manufacture of a reagent kit, wherein the reagent kit is utilized for detecting the presence and / or content of AFP protein.

[0084] In another aspect, the present application provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and / or the modified cell in the manufacture of a medicament for preventing and / or treating tumors.

[0085] In some embodiments, the tumor in the above uses comprises a solid tumor.

[0086] In some embodiments, the tumor in the above use comprises a non-solid tumor.

[0087] In some embodiments, the tumor in the above uses comprises a tumor associated with expression of AFP.

[0088] In some embodiments, the tumor in the above use comprises liver cancer.

[0089] In another aspect, the present application provides a use of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and / or the modified cell for preventing and / or treating a tumor.

[0090] In some embodiments, the tumor in the above uses comprises a solid tumor.

[0091] In some embodiments, the tumor in the above use comprises a non-solid tumor.

[0092] In some embodiments, the tumor in the above uses comprises a tumor associated with expression of AFP.

[0093] In some embodiments, the tumor in the above use comprises liver cancer.

[0094] In another aspect, the present application provides a method for preventing and / or treating a disease or condition, comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and / or the modified cell.

[0095] In some embodiments, the tumor in the above methods comprises a solid tumor.

[0096] In some embodiments, the tumor in the above methods comprises a non-solid tumor.

[0097] In some embodiments, the tumor in the above methods comprises a tumor associated with expression of AFP.

[0098] In some embodiments, the tumor in the above methods comprises liver cancer.

[0099] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. It will be apparent to those skilled in the art that the present application may enable them to modify the specific embodiments disclosed without departing from the spirit and scope of the present invention. Accordingly, the drawings and description herein are merely illustrative and not limiting. [Brief explanation of the drawings]

[0100] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows. [Figure 1] ELISA identification results of five rounds of panning described in this application. [Figure 2] 1 shows the sequence repeatability of monoclonals obtained after panning as described in this application. [Figure 3] 1 shows the results of PCR amplification of candidate VHH antibody nucleotide sequences described in this application. [Figure 4] 1 shows the results of SDS-PAGE of VHH antibodies after expression and purification as described in this application. [Figure 5] 1 shows the eukaryotic expression vectors of the candidate VHH-Fc antibodies described in this application (1C11 is used as an example). [Figure 6] 1 shows the affinity of candidate VHH-Fc antibodies described in this application. [Figure 7A] 1 shows the results of flow cytometry detection of candidate VHH-Fc antibodies described in the present application and human TERT540 / T2 cells. [Figure 7B] 1 shows the results of flow cytometry detection of candidate VHH-Fc antibodies and polypeptides / T2 cells described in the present application. [Figure 7C] 1 shows the results of flow cytometry detection of candidate VHH-Fc antibodies described in the present application and mouse AFP158 / T2 cells. [Figure 8] 1 shows the core plasmid vector profile described in this application. [Figure 9] 1 shows a diagram of the CAR core plasmid structure described in this application. [Figure 10A] 1 shows the results of PCR amplification of the human AFP158-166 nucleotide sequence described in this application. [Figure 10B] 1 shows the results of enzymatic cleavage of the vector described in this application. [Figure 11] 1 shows the results of the flow cytometry screening detection of monoclonal antibodies described in this application. [Figure 12] 1 shows the results of screening detection of the positive rates of HEPG2-MiniG and SK-HEP-1-MiniG by flow cytometry as described in the present application. [Figures 13A-13B] 1 shows the results of a repeated stimulation and expansion experiment of CAR-T cells described in the present application. [Figure 13C] 1 shows the results of an in vitro cell killing experiment of CAR-T cells with an ET ratio of 1:1, as described herein. [Figure 13D] 1 shows the results of IFN-γ cytokine secretion with an ET ratio of 1:1 as described in this application. [Figure 13E] 1 shows the results of IL-2 cytokine secretion with an ET ratio of 1:1 as described in this application. [Figure 13F] 1 shows the results of an in vitro cell killing experiment of CAR-T cells with an ET ratio of 1:1, as described herein. [Figure 13G] 1 shows the results of IFN-γ cytokine secretion with an ET ratio of 1:1 as described in this application. [Figure 13H] 1 shows the results of IL-2 cytokine secretion with an ET ratio of 1:1 as described in this application. [Figure 14] 1 shows a flow chart of the animal experiments described in this application. [Figure 15A] 1 shows a tumor growth graph as described in the present application. [Figure 15B] 1 shows a graph of animal weights described in the present application. DETAILED DESCRIPTION OF THE INVENTION

[0101] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0102] Definition of Terms As used herein, the term "isolated antigen-binding protein" generally refers to a polypeptide polymer that can specifically recognize and / or neutralize a particular antigen. For example, the isolated antigen-binding protein may include a portion of a heavy chain. For example, the isolated antigen-binding protein may include a heavy chain variable region. The term "isolated antigen-binding protein" may also include single domain antibodies. For example, the isolated antigen-binding protein may include, but is not limited to, a human single domain antibody.

[0103] As used herein, the term "single domain antibody" or "sdAb" or "VHH" generally refers to a type of antibody lacking the antibody light chain but possessing only a heavy chain variable region. In some cases, single domain antibodies can be derived from Bactrian camel, dromedary, alpaca, llama, nurse shark, astragalus, or shark (see, for example, Kang Xiao-Sen et al., Biotechnology Reports, 2018, 34(12):1974-1984). For example, a single domain antibody can be derived from an alpaca. A single domain antibody can be composed of a heavy chain variable region (VH). The term "heavy chain variable region" generally refers to the amino-terminal domain of the heavy chain of an antigen-binding fragment. The heavy chain variable region may be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more highly conserved regions known as framework regions (FRs). Each heavy chain variable region is composed of three CDR and four FR regions, which may be arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The heavy chain variable region contains a binding domain that interacts with an antigen (e.g., AFP).

[0104] In this application, the term "TCR-like antibody" generally refers to an antibody that can recognize a peptide / MHC complex on the surface of a tumor cell. These TCR-like antibodies share functional identity with TCRs in terms of target recognition. Technically, TCR-like antibodies can be produced by conventional hybridoma techniques or in vitro antibody library techniques known to those skilled in the art. In this application, the "TCR-like antibody" can recognize AFP / HLA02. For example, the TCR-like antibody can recognize human AFP / HLA02. 158-166 / Can recognize the HLA-A02*01* complex.

[0105] In this application, the term "transmembrane domain" generally refers to a segment of a cell surface protein that spans the cell membrane and may contain a hydrophobic alpha helix. The transmembrane domain can be linked to an intracellular signaling domain and plays a role in signal transduction. In this application, the transmembrane domain can be derived from any type I, type II, or type III transmembrane protein. In the present application, the transmembrane domain may comprise a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154, and SLAM, or a combination thereof. For example, the transmembrane domain may comprise a transmembrane domain derived from CD8.

[0106] In this application, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising a targeting moiety capable of binding to an antigen and at least one intracellular domain. CAR is a core element of chimeric antigen receptor T cells (CAR-T), and may comprise a targeting moiety (e.g., targeting a tumor-specific antigen and / or a tumor-associated antigen), a signal peptide, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. In this application, the CAR is a fusion protein comprising a targeting moiety capable of binding to an antigen (e.g., AFP) of an antibody. 158-166 Based on the specificity of the HLA-A02*01* complex, CARs can be combined with the activating intracellular domain of the T cell receptor. T cells expressing CARs through genetic engineering can specifically recognize and eliminate malignant cells expressing the target antigen. For a description of CARs and CAR-T cells, see, e.g., Sadelain M, Brentjens R, Rivi `ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013;3(4):388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012;24(5):633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014;257(1):107-126; and WO2013154760, WO2016014789.

[0107] As used herein, the term "costimulatory signaling domain" generally refers to an intracellular domain capable of providing an immune costimulatory molecule, which is a cell surface molecule required for an effective lymphocyte response to an antigen. In some cases, the costimulatory signaling domain may include a costimulatory signaling domain derived from a protein 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, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88, or a combination thereof. For example, the costimulatory signaling domain may include a costimulatory signaling domain derived from 4-1BB.

[0108] As used herein, the term "intracellular signaling domain" generally refers to a domain located inside a cell and capable of transmitting a signal. As used herein, the intracellular signaling domain is capable of transmitting a signal into the cell. Typically, the intracellular signaling domain is any contiguous amino acid sequence for directing protein targeting. In some cases, the intracellular signaling domain may include an intracellular signaling domain derived from a protein selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM, or a combination thereof. For example, the intracellular signaling domain may include an intracellular signaling domain derived from CD3ζ.

[0109] As used herein, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. For example, an antibody may include an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or any molecule containing an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments, or antibody derivatives, including, but not limited to, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), and antigen-binding antibody fragments (e.g., Fab, Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, aglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to the antigens described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions may be further divided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FR regions, which may be arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including multiple types of cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0110] In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length antibody fragment. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab', or F(ab')2, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab', or F(ab')2. (1) Fab fragments, which are typically monovalent fragments consisting of the VL, VH, CL, and CH domains; (2) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds at the hinge region; (3) Fd fragments consisting of the VH and CH domains; (4) Fv fragments consisting of the VL and VH domains of a single antibody arm; (5) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) combinations of two or more isolated CDRs, optionally linked by a linker. It may also include a monovalent single-chain molecule, Fv (scFv), formed from a pair of VL and VH (see Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883). The above-mentioned "antigen-binding fragment" may further include an immunoglobulin fusion protein containing a binding domain selected from (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (2) an immunoglobulin heavy chain CH2 constant region fused to a hinge region, and (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. For example, the above-mentioned antigen-binding fragment may further include a single-domain antibody.

[0111] As used herein, the term "monoclonal antibody" generally refers to a population of substantially homogeneous antibodies, i.e., each antibody in the population is homologous except for minor naturally occurring mutations. Monoclonal antibodies are highly specific and are directed against a single antigenic site. For example, such monoclonal antibodies may be produced by hybridoma technology or may be produced in bacterial, eukaryotic, or plant cells using recombinant DNA methods. Monoclonal antibodies may be obtained from phage antibody libraries and produced using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0112] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence of an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequence of another species. For example, the variable regions of the light and heavy chains are derived from the variable regions of an antibody of one animal species (e.g., mouse, rat, etc.), while the constant regions are homologous to antibody sequences derived from another species (e.g., human). For example, to obtain a chimeric antibody, the variable regions may be produced by non-human B cells or hybridoma cells, but the associated constant regions are derived from humans. Such variable regions have the advantage of being easily produced, and their specificity is not affected by the origin of the associated constant regions. At the same time, because the constant regions of chimeric antibodies are derived from humans, the possibility of an immune response caused by a chimeric antibody upon injection is lower than when antibodies with non-human constant regions are used.

[0113] As used herein, the term "humanized antibody" generally refers to a chimeric antibody that has reduced sequences derived from non-human immunoglobulin, reducing the immunogenicity of heterologous antibodies when introduced into humans, while retaining the full antigen-binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and its variants have been used to perform "reshaping" (Verhoeyen et al., 1988 Science 239:1534-1536; Riechmann et al., 1988 Nature 332:323-337; Tempest et al., Bio / Technol 1991 9:266-271) and "hyperchimerization" (Queen et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co et al., 1992 J Immunol Humanization of non-human binding domains can be achieved by techniques such as "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), and resurfacing (U.S. Patent No. US5639641). For example, if other regions, such as the hinge region and constant region domains, are of non-human origin, these regions can also be humanized.

[0114] In the present application, the term "ribosome hopping site", also known as an internal ribosome entry site (IRES), refers to a single nucleotide sequence typically located in the middle of an mRNA sequence and providing for translation initiation. The ribosome hopping site allows translation to be initiated in a cap-independent manner. The IRES is typically located in the 5'UTR. In the present application, the ribosome hopping site may comprise the sequence from 1 to 578 in SEQ ID NO: 145.

[0115] As used herein, the term "tumor" generally refers to a neoplasm formed by local tissue cell proliferation. For example, the tumor may include a solid tumor. For example, the tumor may include a tumor associated with AFP expression. The term "tumor associated with AFP expression" generally refers to altered AFP expression in the tumor microenvironment or tumor compared to normal cells. For example, the "tumor associated with AFP expression" may be a tumor in which the expression level of AFP is upregulated in the tumor microenvironment or tumor compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In an AFP-positive tumor, the protein expression level of AFP in tumor cells or the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than in normal cells.

[0116] In this application, the term "immunoconjugate" generally refers to a complex formed by conjugating (e.g., covalently binding via a linker) the other therapeutic agent with the isolated antigen-binding protein, and the complex specifically binds to an antigen in a target cell via the isolated antigen-binding protein, thereby delivering the other therapeutic agent to the target cell (e.g., a tumor cell). Alternatively, the antigen may be secreted from the target cell and located in the extracellular space of the target cell.

[0117] In this application, "K D " (similarly, "K D " or "K D") usually refers to the "affinity constant" or "equilibrium dissociation constant", and further refers to the value obtained by dividing the equilibrium or dissociation rate constant (kd) by the binding rate constant (ka) in a titration measurement. D ) indicates the binding affinity of a binding protein (e.g., an isolated antigen-binding protein described herein) to an antigen (e.g., an AFP protein). Methods for determining association and dissociation rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and the ability to detect samples at equilibrium in physiological buffers. For example, the above K can be determined by Biacore (Biomolecular Interaction Analysis) (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). D Other experimental routes and instruments, such as Octet detection, are available to measure K values. Alternatively, the K values ​​can be measured by KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho). D The K value can be measured or determined by surface plasmon resonance (SPR). D The value can be measured.

[0118] As used herein, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length in isolated form, either isolated from their natural environment or artificially synthesized.

[0119] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of autonomous replication in a suitable host. Such vectors can transfer inserted nucleic acid molecules within cells and / or between cells. Such vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for expressing transcription and / or translation of DNA or RNA. Such vectors may be polynucleotides that can be transcribed and translated into a polypeptide when introduced into an appropriate cell. Typically, by culturing appropriate cells containing such vectors, such vectors can produce the desired expression product. As used herein, such vectors may include lentiviral vectors.

[0120] As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or has contained a plasmid or vector containing a nucleic acid molecule described herein, or that is capable of expressing a chimeric antigen receptor or antigen-binding protein described herein. Such a cell may include the progeny of a single cell. Due to natural, unexpected, or deliberate mutations, progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may still express a chimeric antigen receptor or antigen-binding protein described herein. Such cells may be obtained by transfecting cells in vitro with a vector described herein. Such cells may be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, such cells may be immune cells. For example, the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells. For example, the immune cells may be T cells.

[0121] As used herein, the term "treatment" generally refers to (i) preventing the disease, disease, and / or condition in a patient who is susceptible to, but has not yet been diagnosed with, the disease, disease, or condition; (ii) inhibiting the disease, disease, or condition, i.e., slowing its progression; and (iii) palliating the disease, disease, or condition, i.e., reducing the disease, disease, and / or condition and / or the symptoms associated with the disease, disease, and / or condition.

[0122] As used herein, the terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also cover polymers of modified amino acids. These modifications may include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (e.g., conjugation with a labeling component). The term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptidomimetics.

[0123] As used herein, the terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple loci (single locus) analyzed based on linkage, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after incorporation into the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0124] Except for the specific proteins and nucleotides mentioned herein, the present application may further include functional variants, derivatives, analogs, homologs, and fragments thereof.

[0125] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence or is encoded by a substantially identical nucleotide sequence and that can possess one or more activities of the naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that said polypeptide or polynucleotide essentially retains at least one intrinsic function. Variant sequences can be obtained by adding, deleting, substituting, modifying, exchanging, and / or mutating at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide, so long as the original functional activity is retained.

[0126] As used herein, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that includes any substitution, mutation, modification, exchange, deletion, and / or addition of one or more amino acid residues of the self / paired sequence, so long as the resulting polypeptide or polynucleotide essentially retains at least one of its endogenous functions.

[0127] As used herein, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0128] Generally, amino acid substitutions can be made, e.g., at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence retains essentially the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0129] Proteins or polypeptides used herein can have deletions, insertions, or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties of the residues, so long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids containing uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0130] In this application, the term "AFP antigen" generally refers to a glycoprotein belonging to the albumin family. The amino acid sequence of human AFP is shown in UniProt / Swiss-Prot accession number P02771.

[0131] As used herein, the term "T cells," also known as T lymphocytes, refers to a type of white blood cell isoform that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by T cell receptors present on the cell surface. As used herein, T cells may include stem cell-like memory T cells (TSCM) and central memory T cells (TCM).

[0132] In this application, the term "T cell receptor," commonly referred to as "TCR," generally refers to the molecular structure of a T cell that specifically recognizes and binds to an antigen peptide-MHC molecule. The T cell receptor can be present on the surface of a T cell in a complex with a CD3 molecule. The TCR may be a heterodimer anchored in the cell membrane, with the majority of the heterodimer consisting of highly variable α and β subunits linked by disulfide bonds, and the minority of the heterodimer consisting of γ and δ peptides. The TCR may comprise a variable region and a constant region, the constant region being adjacent to the cell membrane and connecting the transmembrane region to the intracellular end, and the variable region recognizing the polypeptide / MHC complex.

[0133] As used herein, the term "low-density lipoprotein receptor-related protein" (LRP) generally refers to an endogenous protein containing 839 amino acids (excluding a 21-amino acid signal peptide). It is embedded in the outer phospholipid layer of low-density lipoprotein (LDL) particles, belongs to an endocytic receptor, and can mediate the endocytosis of cholesterol-rich LDL. It is a member of the low-density lipoprotein receptor (LDLR) gene family. Expression of LRP is most prominent in bronchial epithelial cells, adrenal gland, and cortical tissue. As used herein, the low-density lipoprotein receptor-related protein may include one or more proteins selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and truncations thereof.

[0134] In this application, the terms "low-density lipoprotein receptor-related protein 6" (LRP-6) and "low-density lipoprotein receptor-related protein 5" (LRP-5) generally refer to distinct subgroups of the low-density lipoprotein receptor (LDLR) family. Human LRP-6 has UniProt accession number O75581. Human LRP-5 has UniProt accession number O75197.

[0135] As used herein, the term "truncated protein" generally refers to a truncated protein. Such truncated proteins can be obtained by removing the N- or C-terminal portions of a protein through proteolysis or by engineering the structural gene. Alternatively, such truncated proteins can be obtained by prematurely terminating translation by introducing a stop codon into the structural gene through a nonsense mutation.

[0136] In this application, the term "and / or" should be understood to mean either one of the alternatives or both of the alternatives.

[0137] In this application, the term "comprising" generally means including the explicitly specified features but not excluding other elements.

[0138] In this application, the term "about" generally refers to a variation of 0.5% to 10% or more or less from the specified value, for example, a variation 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% or more or less from the specified value.

[0139] In this application, the term "comprising" generally means including, grouping together, containing or encompassing. In some contexts, it also means "is" or "consists of." Detailed Description of the Invention

[0140] The isolated antigen-binding proteins described herein In one aspect, the present application provides an isolated antigen binding protein, which has a K of 3.1E-09 M or less as measured by a ForteBio AHC sensor. D value (e.g., the above K D less than about 3.1E-09 M, less than about 3.0E-09 M, less than about 2.9E-09 M, less than about 2.8E-09 M, less than about 2.7E-09 M, less than about 2.6E-09 M, less than about 2.7E-09 M, less than about 2.6E-09 M, less than about 2.3E-09 M, less than about 2.0E-09 M, less than about 1.5E-09 M, less than about 1E-09 M, or less than 5E-10 M or less) of human AFP 158-166 / Can specifically bind to the HLA-A02*01* complex.

[0141] In one aspect, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR of an antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 72 to SEQ ID NO: 84.

[0142] In the present application, the HCDR of the isolated antigen-binding protein may be split in any form, and as long as the VH is the same as the amino acid sequence shown in SEQ ID NO: 72 to SEQ ID NO: 84, the HCDR obtained by splitting in any form is included in the scope of protection of the present application.

[0143] The CDR of an antibody, also known as the complementarity-determining region, is a part of the variable region. The amino acid residues in this region can contact an antigen or antigen epitope. Antibody CDRs can be determined using various numbering systems, such as CCG, Kabat, Chothia, IMGT, AbM, North's, or, when considered comprehensively, Kabat / Chothia. These numbering systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine CDR regions using different numbering systems depending on the antibody sequence and structure. Using different numbering systems may result in differences in the CDR regions. In this application, the term "CDR" refers to CDR sequences divided according to any CDR division scheme, as well as variants thereof, which include substitutions, deletions, and / or additions of one or more amino acids in the amino acid sequence of the CDR. For example, the substitution, deletion, and / or insertion of 1 to 30, 1 to 20, or 1 to 10, or further for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids is covered, and homologs thereof are also covered. The homologs may be amino acid sequences that have at least about 85% sequence homology (e.g., 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) with the amino acid sequence of the CDR.

[0144] In the present application, the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.

[0145] In the present application, the HCDR3 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR3 sequence of the antigen-binding protein may be defined according to the IMGT numbering system.

[0146] In the present application, the HCDR3 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56. For example, the HCDR3 sequence of the antigen-binding protein may be defined according to the Kabat numbering system.

[0147] In the present application, the HCDR2 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53. For example, the HCDR2 sequence of the antigen-binding protein may be defined according to the IMGT numbering system.

[0148] In the present application, the HCDR2 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the antigen-binding protein may be defined by the Kabat numbering system.

[0149] In the present application, the HCDR1 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57. For example, the HCDR1 sequence of the antigen-binding protein may be defined according to the IMGT numbering system.

[0150] In the present application, the HCDR1 of the antigen-binding protein may comprise any one of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the antigen-binding protein may be defined by the Kabat numbering system.

[0151] For example, HCDR1 of the antigen-binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57, the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53, and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR1, HCDR2, and HCDR3 sequences of the antigen-binding protein may be defined according to the IMGT numbering system.

[0152] In the present application, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50. For example, the HCDR1, HCDR2, and HCDR3 sequences of the antigen binding protein can be defined by the Kabat numbering system.

[0153] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, the antigen binding protein may comprise antibody 1B3 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1B3.

[0154] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the antigen binding protein may comprise antibody 1C4 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1C4.

[0155] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the antigen binding protein may comprise antibody 1C11 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1C11.

[0156] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the antigen binding protein may comprise antibody 1D12 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1D12.

[0157] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the antigen binding protein may comprise antibody 2F9 or an antigen-binding fragment having the same HCDR3 (e.g. having the same HCDR1-3) as antibody 2F9.

[0158] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the antigen binding protein may comprise antibody no. 3 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0159] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 39, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the antigen binding protein may comprise antibody No. 4 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0160] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the antigen binding protein may comprise antibody no. 7 or an antigen-binding fragment having the same HCDR3 (e.g. having the same HCDR1-3) as that of antibody no.

[0161] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the antigen binding protein may comprise antibody no. 8 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0162] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 44, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 46. For example, the antigen binding protein may comprise antibody no. 15 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0163] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 50, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the antigen binding protein may comprise antibody no. 17 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0164] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56. For example, the antigen binding protein may comprise antibody no. 24 or an antigen-binding fragment having the same HCDR3 therein (e.g. having the same HCDR1-3 therein).

[0165] For example, HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the antigen binding protein may comprise antibody no. 29 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no.

[0166] For example, the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0167] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:65.

[0168] In the present application, the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:66.

[0169] In the present application, H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69.

[0170] In the present application, H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0171] In the present application, the H-FR1 of the antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 58 to 65, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 66, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 67 to 69, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 70 to 71.

[0172] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 58, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody 1B3 or an antigen-binding fragment thereof having the same H-FR1-4.

[0173] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 59, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody 1C4 or an antigen-binding fragment thereof having the same H-FR1-4.

[0174] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 60, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody 1C11 or an antigen-binding fragment thereof having the same H-FR1-4.

[0175] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 60, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody 1D12 or an antigen-binding fragment thereof having the same H-FR1-4.

[0176] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody 2F9 or an antigen-binding fragment thereof having the same H-FR1-4.

[0177] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 59, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody No. 3 or an antigen-binding fragment having the same H-FR1-4.

[0178] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 62, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody No. 4 or an antigen-binding fragment having the same H-FR1-4.

[0179] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 60, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody No. 7 or an antigen-binding fragment having the same H-FR1-4.

[0180] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 63, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody No. 8 or an antigen-binding fragment having the same H-FR1-4.

[0181] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 64, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody no. 15 or an antigen-binding fragment having the same H-FR1-4.

[0182] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 64, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 68, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 71. For example, the antigen-binding protein may comprise antibody no. 17 or an antigen-binding fragment having the same H-FR1-4.

[0183] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 61, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody no. 24 or an antigen-binding fragment having the same H-FR1-4.

[0184] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 65, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 69, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the antigen-binding protein may comprise antibody no. 29 or an antigen-binding fragment having the same H-FR1-4.

[0185] In the present application, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 72 to 84.

[0186] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 58, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 72. For example, the antigen-binding protein may comprise antibody 1B3 or an antigen-binding protein having the same heavy chain variable region therewith.

[0187] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 73. For example, the antigen-binding protein may comprise antibody 1C4 or an antigen-binding protein having the same heavy chain variable region therewith.

[0188] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 74. For example, the antigen-binding protein may comprise antibody 1C11 or an antigen-binding protein having the same heavy chain variable region therewith.

[0189] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:29, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO:75. For example, the antigen-binding protein may comprise antibody 1D12 or an antigen-binding protein having the same heavy chain variable region therewith.

[0190] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 76. For example, the antigen-binding protein may comprise antibody 2F9 or an antigen-binding protein having the same heavy chain variable region therewith.

[0191] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 77. For example, the antigen-binding protein may comprise antibody No. 3 or an antigen-binding protein having the same heavy chain variable region therewith.

[0192] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 39, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 62, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 78. For example, the antigen-binding protein may comprise antibody No. 4 or an antigen-binding protein having the same heavy chain variable region therewith.

[0193] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 79. For example, the antigen-binding protein may comprise antibody No. 7 or an antigen-binding protein having the same heavy chain variable region therewith.

[0194] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 63, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 80. For example, the antigen-binding protein may comprise antibody No. 8 or an antigen-binding protein having the same heavy chain variable region therewith.

[0195] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 44, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 46. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 81. For example, the antigen-binding protein may comprise antibody No. 15 or an antigen-binding protein having the same heavy chain variable region therewith.

[0196] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 50, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 68, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 71. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 82. For example, the antigen-binding protein may comprise antibody no. 17 or an antigen-binding protein having the same heavy chain variable region therewith.

[0197] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 83. For example, the antigen-binding protein may comprise antibody No. 24 or an antigen-binding protein having the same heavy chain variable region therewith.

[0198] In the present application, the antigen-binding protein may comprise a heavy chain variable region, which may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 65, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 69, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 84. For example, the antigen-binding protein may comprise antibody no. 29 or an antigen-binding protein having the same heavy chain variable region therewith.

[0199] In the present application, the isolated antigen-binding protein is human AFP. 158-166 / HLA-A02*01* complex can compete with a reference antibody for binding.

[0200] In the present application, the reference antibody may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.

[0201] In the present application, the HCDR3 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR3 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0202] In the present application, the HCDR3 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56. For example, the HCDR3 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0203] In the present application, the HCDR2 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53. For example, the HCDR2 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0204] In the present application, the HCDR2 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0205] In the present application, the HCDR1 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57. For example, the HCDR1 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0206] In the present application, the HCDR1 of the reference antibody may comprise any one of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0207] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57, the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53, and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR1, HCDR2, and HCDR3 sequences of the reference antibody may be defined according to the IMGT numbering system.

[0208] In the present application, the reference antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50. For example, the HCDR1, HCDR2, and HCDR3 sequences of the reference antibody can be defined according to the Kabat numbering system.

[0209] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, the reference antibody may comprise antibody 1B3 or an antigen-binding fragment having the same HCDR3 as antibody 1B3 (e.g., having the same HCDR1-3 as antibody 1B3).

[0210] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the reference antibody may comprise antibody 1C4 or an antigen-binding fragment having the same HCDR3 as that (e.g., having the same HCDR1-3 as that).

[0211] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the reference antibody may comprise antibody 1C11 or an antigen-binding fragment having the same HCDR3 as that (e.g., having the same HCDR1-3 as that).

[0212] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the reference antibody may comprise antibody 1D12 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1D12.

[0213] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the reference antibody may comprise antibody 2F9 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as that of antibody 2F9.

[0214] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the reference antibody may comprise antibody No. 3 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody No. 3.

[0215] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 39, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the reference antibody may comprise antibody No. 4 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody No. 4.

[0216] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the reference antibody may comprise antibody No. 7 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody No. 7.

[0217] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the reference antibody may comprise antibody No. 8 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody No. 8.

[0218] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 44, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 46. For example, the reference antibody may comprise antibody No. 15 or an antigen-binding fragment having the same HCDR3 as that (e.g., having the same HCDR1-3 as that).

[0219] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 50, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the reference antibody may comprise antibody No. 17 or an antigen-binding fragment having the same HCDR3 as that (e.g., having the same HCDR1-3 as that).

[0220] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56. For example, the reference antibody may comprise antibody No. 24 or an antigen-binding fragment having the same HCDR3 as that (e.g., having the same HCDR1-3 as that).

[0221] For example, HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the reference antibody may comprise antibody No. 29 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody No. 29.

[0222] Chimeric antigen receptors and modified cells In the present application, the targeting portion of the chimeric antigen receptor may comprise a heavy chain variable region VH, and the VH may comprise at least one, two, or three of HCDR1, HCDR2, and HCDR3.

[0223] In the present application, the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.

[0224] In the present application, the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 52, and SEQ ID NO: 56. For example, the HCDR3 sequence of the targeting portion of the chimeric antigen receptor can be defined by the Kabat numbering system.

[0225] In the present application, the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined by the IMGT numbering system.

[0226] In the present application, the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 55. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor can be defined by the Kabat numbering system.

[0227] In the present application, the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57. For example, the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined according to the IMGT numbering system.

[0228] In the present application, the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences set forth in SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO: 50. For example, the HCDR1 sequence of the targeting portion of the chimeric antigen receptor can be defined by the Kabat numbering system.

[0229] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in any one of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 57, the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 53, and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 49, and SEQ ID NO: 54. For example, the HCDR1, HCDR2, and HCDR3 sequences of the targeting portion of the chimeric antigen receptor may be defined according to the IMGT numbering system.

[0230] In the present application, the targeting portion of the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 19, 25, 30, 35, 46, 52, and 56, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 24, 34, 45, 51, and 55, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17, 23, 29, 37, 39, 44, and 50. For example, the HCDR1, HCDR2, and HCDR3 sequences of the targeting portion of the chimeric antigen receptor can be defined by the Kabat numbering system.

[0231] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1B3 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1B3.

[0232] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1C4 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1C4.

[0233] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1C11 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1C11.

[0234] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1D12 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 1D12.

[0235] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 2F9 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody 2F9.

[0236] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 3 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 3.

[0237] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 39, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 4 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 4.

[0238] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 7 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 7.

[0239] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 8 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as that.

[0240] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 44, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 46. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 15 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 15.

[0241] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 50, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 17 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 17.

[0242] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 24 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 24.

[0243] For example, HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the targeting portion of the chimeric antigen receptor may comprise antibody no. 29 or an antigen-binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) as antibody no. 29.

[0244] For example, the VH of the targeting portion of the chimeric antigen receptor may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0245] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences shown in SEQ ID NO:58 to SEQ ID NO:65.

[0246] In the present application, the H-FR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:66.

[0247] In the present application, H-FR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69.

[0248] In the present application, H-FR4 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0249] In the present application, the H-FR1 of the target portion of the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NOs: 58 to 65, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 66, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 67 to 69, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 70 to 71.

[0250] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 58, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1B3 or an antigen-binding fragment thereof having the same H-FR1-4.

[0251] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1C4 or an antigen-binding fragment having the same H-FR1-4.

[0252] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1C11 or an antigen-binding fragment thereof having the same H-FR1-4.

[0253] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 1D12 or an antigen-binding fragment thereof having the same H-FR1-4.

[0254] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody 2F9 or an antigen-binding fragment thereof having the same H-FR1-4.

[0255] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 3 or an antigen-binding fragment having the same H-FR1-4.

[0256] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 62, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 4 or an antigen-binding fragment having the same H-FR1-4.

[0257] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 7 or an antigen-binding fragment having the same H-FR1-4.

[0258] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 63, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 8 or an antigen-binding fragment having the same H-FR1-4.

[0259] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 15 or an antigen-binding fragment having the same H-FR1-4.

[0260] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 68, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 71. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 17 or an antigen-binding fragment having the same H-FR1-4.

[0261] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 24 or an antigen-binding fragment having the same H-FR1-4.

[0262] In the present application, H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 65, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 69, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the targeting portion of the chimeric antigen receptor may comprise antibody No. 29 or an antigen-binding fragment having the same H-FR1-4.

[0263] In the present application, the heavy chain variable region of the targeting portion of the chimeric antigen receptor may comprise any one of the amino acid sequences shown in SEQ ID NO:72 to SEQ ID NO:84.

[0264] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 58, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 72. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody 1B3 or a chimeric antigen receptor having the same heavy chain variable region as antibody 1B3.

[0265] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 73. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody 1C4 or a chimeric antigen receptor having the same heavy chain variable region as antibody 1C4.

[0266] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 74. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody 1C11 or a chimeric antigen receptor having the same heavy chain variable region as antibody 1C11.

[0267] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:29, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO:75. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody 1D12 or a chimeric antigen receptor having the same heavy chain variable region as antibody 1D12.

[0268] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 76. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody 2F9 or a chimeric antigen receptor having the same heavy chain variable region as antibody 2F9.

[0269] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 59, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 77. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 3 or a chimeric antigen receptor having the same heavy chain variable region therewith.

[0270] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 39, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 62, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 78. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 4 or a chimeric antigen receptor having the same heavy chain variable region.

[0271] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 60, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 79. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 7 or a chimeric antigen receptor having the same heavy chain variable region.

[0272] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 63, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 80. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 8 or a chimeric antigen receptor having the same heavy chain variable region therewith.

[0273] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 44, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 46. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 81. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 15 or a chimeric antigen receptor having the same heavy chain variable region as antibody No. 15.

[0274] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 50, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 64, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 68, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 71. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 82. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 17 or a chimeric antigen receptor having the same heavy chain variable region.

[0275] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 56. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 61, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 67, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 83. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 24 or a chimeric antigen receptor having the same heavy chain variable region.

[0276] In the present application, the targeting moiety of the chimeric antigen receptor may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 30. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 65, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 69, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 70. For example, the heavy chain variable region of the chimeric antigen receptor targeting moiety may comprise the amino acid sequence set forth in SEQ ID NO: 84. For example, the targeting moiety of the chimeric antigen receptor may comprise the targeting moiety of antibody No. 29 or a chimeric antigen receptor having the same heavy chain variable region.

[0277] In the present application, the chimeric antigen receptor may comprise a hinge region. For example, the hinge region may comprise a hinge region derived from the following proteins: IgG4, IgG1, and CD8. For example, the hinge region may comprise the amino acid sequence set forth in SEQ ID NO: 147. For example, the hinge region may comprise an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID NO: 147. For example, the nucleotide sequence encoding the hinge region may comprise the nucleotide sequence set forth in SEQ ID NO: 148.

[0278] In the present application, the chimeric antigen receptor may comprise a transmembrane domain. For example, the transmembrane domain may comprise, but is not limited to, a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154, and SLAM, or a combination thereof. For example, the transmembrane domain may comprise a transmembrane domain derived from CD8. For example, the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO: 149. For example, the transmembrane domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 149. For example, the nucleotide sequence encoding the transmembrane domain may comprise the nucleotide sequence set forth in SEQ ID NO: 150.

[0279] For example, the hinge and transmembrane regions may comprise the amino acid sequence set forth in SEQ ID NO: 128. For example, the hinge and transmembrane regions may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 128. For example, the hinge and transmembrane regions may comprise the nucleotide sequence set forth in SEQ ID NO: 127.

[0280] In the present application, the chimeric antigen receptor may comprise a costimulatory signaling domain. For example, the costimulatory signaling domain may comprise, but is not limited to, a costimulatory signaling domain derived from a protein 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, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88, or a combination thereof. For example, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from 4-1BB. For example, the costimulatory signaling domain may comprise the amino acid sequence set forth in SEQ ID NO: 132. For example, the costimulatory signaling domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 132. For example, the nucleotide sequence encoding the costimulatory signaling domain can comprise the nucleotide sequence of SEQ ID NO: 131.

[0281] For example, the costimulatory signaling domain may comprise a costimulatory signaling domain derived from CD28. For example, the costimulatory signaling domain may comprise the amino acid sequence set forth in SEQ ID NO: 130. For example, the costimulatory signaling domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 130. For example, the nucleotide sequence encoding the costimulatory signaling domain may comprise the nucleotide sequence of SEQ ID NO: 129.

[0282] In the present application, the chimeric antigen receptor may comprise an intracellular signaling domain. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from a protein selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, and DAP-12, or a combination thereof. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from CD3ζ. For example, the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO: 134. For example, the intracellular signaling domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 134. For example, the nucleotide sequence encoding the intracellular signaling domain can comprise the nucleotide sequence of SEQ ID NO: 133.

[0283] For example, the N-terminus of the transmembrane domain may be linked to the C-terminus of the targeting moiety. For example, the C-terminus of the transmembrane domain may be linked to the N-terminus of the costimulatory signaling domain. For example, the C-terminus of the costimulatory signaling domain may be linked to the N-terminus of the intracellular signaling domain.

[0284] For example, the chimeric antigen receptor may comprise the following domains in order from N-terminus to C-terminus: a targeting moiety, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0285] For example, the chimeric antigen receptor may comprise the following domains in order from the N-terminus to the C-terminus: an antigen-binding protein of the present application (e.g., a VHH), a transmembrane domain derived from CD8, a costimulatory signaling domain derived from CD137, and an intracellular signaling domain derived from CD3zeta.

[0286] In the present application, the chimeric antigen receptor may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 112 to 124. For example, the chimeric antigen receptor may comprise an amino acid sequence that has at least 80% sequence identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID NOs: 112 to 124.

[0287] In another aspect, the present application provides a modified cell, wherein the modified cell may comprise the chimeric antigen receptor. In the present application, the modification may comprise upregulating the expression level of a low-density lipoprotein receptor-related protein or a fragment thereof in the cell.

[0288] Low density lipoprotein receptor-related protein or a fragment thereof In the present application, the low-density lipoprotein receptor-related protein may include one or more proteins selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

[0289] In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof may be derived from a mammal, such as a human, a macaque, a rat, or a mouse. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be derived from a human.

[0290] In the present application, the functional fragment may include a fragment or truncation of the low-density lipoprotein receptor-related protein having the activity of the low-density lipoprotein receptor-related protein. For example, the low-density lipoprotein receptor-related protein may include low-density lipoprotein receptor-related protein 6 and truncations thereof, and / or low-density lipoprotein receptor-related protein 5 and truncations thereof.

[0291] In the present application, the truncated form of low-density lipoprotein receptor-related protein 6 may comprise the intracellular region of low-density lipoprotein receptor-related protein 6, and / or the truncated form of low-density lipoprotein receptor-related protein 5 may comprise the intracellular region of low-density lipoprotein receptor-related protein 5. In the present application, the term "intracellular region" generally refers to a domain of a protein located within a cell membrane. In the present application, the intracellular region may refer to a domain within the cell membrane of the low-density lipoprotein receptor-related protein. In the present application, the intracellular region may comprise the sequence of amino acids 24 to 243 in SEQ ID NO: 140 or a sequence having at least 80% identity thereto, or may comprise the sequence of amino acids 24 to 231 in SEQ ID NO: 144 or a sequence having at least 80% identity thereto. Furthermore, for example, the truncated form of low-density lipoprotein receptor-related protein 6 may comprise the transmembrane region of low-density lipoprotein receptor-related protein 6 and the LDLR region of low-density lipoprotein receptor-related protein 6, and / or the truncated form of low-density lipoprotein receptor-related protein 5 may comprise the transmembrane region of low-density lipoprotein receptor-related protein 5 and the LDLR region of low-density lipoprotein receptor-related protein 5. In the present application, the term "LDLR region" generally refers to a domain located near the N-terminus outside the transmembrane region of the low-density lipoprotein receptor-related protein. This domain may have a Wnt signal amplification function. In the present application, the LDLR region may comprise the amino acid sequence from positions 5 to 119 of SEQ ID NO: 138 or an amino acid sequence having at least 80% identity thereto, or the sequence from positions 4 to 119 of SEQ ID NO: 142 or an amino acid sequence having at least 80% identity thereto.

[0292] In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, and SEQ ID NO: 144, or an amino acid sequence having at least 80% homology thereto.

[0293] In the present application, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof includes the nucleic acid sequence set forth in any one of SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, and SEQ ID NO: 143, or a nucleic acid sequence having at least 80% homology thereto.

[0294] In the present application, the modified cells may include immune cells (e.g., lymphocytes). In the present application, the modified cells may include modified T cells. In the present application, the modified cells may include modified stem cell-like memory T cells (TSCM) and / or genetically modified central memory T cells (TCM). In the present application, the TSCM may include CCR7+ and / or CD62L+. In the present application, the TSCM further include one or more characteristics selected from the group consisting of CD45RA+ or CD45RA-, CD45RO+ or CD45RO-, CD27+, CD28+, CD127+, CD122+, CD3+, CD4+, and CD8+.

[0295] In the present application, the modified cells are capable of expressing the chimeric antigen receptor (CAR). For example, the modified cells may contain a vector capable of expressing the chimeric antigen receptor (CAR). The vector may contain a nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the vector may be selected from the group consisting of a retroviral vector, a lentiviral vector, and / or a transposon plasmid.

[0296] In the present application, the vector capable of expressing the low-density lipoprotein receptor-related protein or a fragment thereof, and the vector capable of expressing the chimeric antigen receptor (CAR) may be the same vector or different vectors, as long as one or more of these vectors are capable of expressing the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR), thereby allowing the genetically modified immune cells to simultaneously comprise the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR).

[0297] For example, the vector capable of expressing the low-density lipoprotein receptor-related protein or a fragment thereof and the vector capable of expressing the chimeric antigen receptor (CAR) may be the same vector. In the vector, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof and the nucleotide molecule encoding the chimeric antigen receptor (CAR) may be located in the same expression cassette. For example, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the 3' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).

[0298] In the present application, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof may be directly or indirectly linked to the nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the indirect link may be via a linking sequence. The 5' end of the linking sequence may be linked to the 3' end of the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof, and the 3' end of the linking sequence may be linked to the 5' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).

[0299] In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) expressed by the modified immune cells may be two independent proteins. That is, they are not interlinked and form any form of dimer (multimer) or protein complex. However, the low-density lipoprotein receptor-related protein or a fragment thereof and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cells may also be linked to each other. For example, in some cases, the low-density lipoprotein receptor-related protein or a fragment thereof forms a complex with the chimeric antigen receptor because the two proteins formed by translation are not completely cleaved.

[0300] The chimeric antigen receptor may comprise a 2A sequence. The term "2A sequence" generally refers to a single self-cleaving amino acid sequence that is not dependent on a protease. The 2A sequence leads to the production of two proteins by transcription. In the present application, the 2A sequence may comprise the sequence from positions 1 to 54 in SEQ ID NO: 136. For example, the nucleotide sequence encoding the 2A sequence may comprise the nucleotide sequence of SEQ ID NO: 135.

[0301] In the present application, the chimeric antigen receptor may comprise a leader sequence. For example, the nucleotide sequence encoding the leader sequence may comprise the nucleotide sequence of SEQ ID NO: 146.

[0302] Polypeptides and immunoconjugates In another aspect, the present application provides one or more polypeptides, which may comprise the isolated antigen-binding proteins of the present application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide may comprise a multispecific antibody (e.g., a bispecific antibody).

[0303] In another aspect, the present application provides one or more immunoconjugates, which may comprise an isolated antigen-binding protein of the present application. In some embodiments, the immunoconjugates may further comprise a pharmaceutically acceptable therapeutic agent, marker, and / or detection agent.

[0304] Nucleic acids, vectors and cells In another aspect, the present application further provides one or more isolated nucleic acid molecules capable of encoding the isolated antigen-binding proteins or polypeptides described herein. For example, each of the one or more nucleic acid molecules may encode the entire antigen-binding protein or a portion thereof (e.g., HCDR1-3, one or more of the heavy chain variable region).

[0305] For example, where nucleic acid molecules each encode a portion of an antigen binding protein or polypeptide as described above, the products encoded by the nucleic acid molecules can be combined to form a functional (e.g., capable of binding to AFP) isolated antigen binding protein of the present application.

[0306] The nucleic acid molecules described herein may be isolated, e.g., produced by (i) in vitro amplification, e.g., by polymerase chain reaction (PCR), (ii) recombinant production by cloning, (iii) purification, e.g., isolation by enzymatic cleavage and gel electrophoretic fractionation, or (iv) synthesis, e.g., chemical synthesis. For example, the isolated nucleic acid may be a nucleic acid molecule produced by recombinant DNA technology.

[0307] In the present application, nucleic acids encoding the isolated antigen-binding proteins can be produced by several methods known in the art, including, but not limited to, obtaining nucleic acid molecules of the isolated antigen-binding proteins or polypeptides described in the present application by reverse transcription PCR and PCR.

[0308] In another aspect, the present application provides one or more vectors containing one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules. The vector may also contain other genes, such as marker genes, that allow for selection of the vector in an appropriate host cell under appropriate conditions. The vector may also contain expression control elements that enable the correct expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type, but typically includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, capping sequence, or CAAT sequence. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence for transcription and regulation of an operably linked nucleic acid. The expression control sequence may further include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters used for SP6, T3, and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), in which a portion of the promoter can be fused to a portion of a gene promoter for another cellular protein (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain another intron. One or more nucleic acid molecules described in the present application can be operably linked to the above expression control element.

[0309] The vector may include, for example, a plasmid, cosmid, virus, phage, or other vectors commonly used in genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. Viral vectors may be administered to a patient (in vivo) in a direct or indirect manner, for example, by treating cells with the virus in vitro and then administering the treated cells to a patient (ex vivo). Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks on virology and molecular biology. Common virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors used for gene transfer. In some cases, retroviral, lentiviral, and adeno-associated viral methods can be used to transfer and integrate genes into the host genome, allowing for long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce relatively high viral titers. Lentiviral vectors may contain a long terminal repeat (5'LTR), a truncated 3'LTR, an RRE, a rev response element (cPPT), a central termination sequence (CTS), and / or a post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.

[0310] In another aspect, the present application provides cells. The cells may comprise an isolated antigen-binding protein, polypeptide, immunoconjugate, one or more nucleic acid molecules, and / or one or more vectors described herein. For example, each or each cell may comprise one or more nucleic acid molecules or vectors described herein. For example, each or each cell may comprise a plurality (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the host cell, such as a prokaryotic cell (e.g., bacterial cell), CHO cell, NS / 0 cell, HEK293T cell, 293F cell, or HEK293A cell, or other eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell. The vector described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamine transfection. For example, the cell may comprise a yeast cell. For example, the cell may comprise an E. coli cell. For example, the cell may comprise a mammalian cell. For example, the cell may comprise an immune cell.

[0311] The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells. For example, the cells may include T cells.

[0312] Pharmaceutical Composition In another aspect, the present application provides pharmaceutical compositions. The pharmaceutical compositions may comprise the isolated antigen-binding proteins, polypeptides, immunoconjugates, isolated nucleic acid molecules, vectors, or cells described herein, and / or pharmaceutically acceptable adjuvants and / or excipients. Herein, pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low-molecular-weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or non-ionic surfactants. Any conventional vehicle or agent is contemplated for use in the pharmaceutical compositions of the present application, provided it is not incompatible with the cells described herein. Herein, pharmaceutically acceptable excipients may include additives other than the active ingredient in pharmaceutical formulations, and are also referred to as additives. For example, excipients may include binders, fillers, disintegrants, and lubricants in tablets. For example, excipients may include wine, vinegar, or herbal juice in herbal pills. For example, the excipient may include a matrix portion in an ointment or cream, which is a semi-solid preparation. For example, the excipient may include a preservative, antioxidant, flavoring agent, fragrance, cosolvent, emulsifier, solubilizer, osmotic pressure adjuster, or coloring agent in a liquid preparation.

[0313] Reagent Kits, Uses and Methods In another aspect, the present application provides a method for detecting or measuring AFP, said method may comprise using said isolated antigen-binding protein or said polypeptide.

[0314] As used herein, the methods may include in vitro methods, ex vivo methods, non-diagnostic or non-therapeutic methods.

[0315] For example, the method may comprise a method for detecting the presence and / or content of AFP for non-diagnostic purposes, comprising the steps of: 1) contacting a sample with an antigen-binding protein according to the present application; and 2) determining the presence and / or expression level of AFP in a sample obtained from a subject by detecting the presence and / or content of said antigen-binding protein that binds to the sample.

[0316] In another aspect, the present application provides a reagent kit for AFP, which may include the use of the isolated antigen-binding protein or the polypeptide.

[0317] In the present application, the reagent kit may further include instructions describing a method for detecting the presence and / or content of AFP, which may include, for example, an in vitro method, an ex vivo method, a non-diagnostic method, or a non-therapeutic method.

[0318] In another aspect, the present application provides the use of the isolated antigen-binding protein or the polypeptide in the manufacture of a reagent kit, which can be used in a method for detecting the presence and / or content of AFP, which may include, for example, an in vitro method, an ex vivo method, a non-diagnostic method, or a non-therapeutic method.

[0319] In another aspect, the present application provides an isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, or the pharmaceutical composition for preventing, alleviating, and / or treating a disease or condition.

[0320] In another aspect, the reagent kit and / or the pharmaceutical composition of the present application are used for preventing, alleviating and / or treating a disease or condition.

[0321] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with AFP expression. The term "tumor associated with AFP expression" generally refers to altered AFP expression in the tumor microenvironment or tumor cells compared to normal cells. For example, the "tumor associated with AFP expression" may be a tumor with upregulated AFP expression levels in the tumor microenvironment or tumor cells compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In an AFP-positive tumor, the AFP protein expression level in tumor cells or the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or more higher than in normal cells. For example, the tumor may include a solid tumor. For example, the tumor may include liver cancer.

[0322] In another aspect, the present application provides the use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the manufacture of a medicament, wherein the medicament is used to prevent, alleviate and / or treat a disease or condition.

[0323] In another aspect, the present application provides the use of the pharmaceutical composition in the manufacture of a medicament, wherein the medicament is used to prevent, alleviate and / or treat a disease or condition.

[0324] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with AFP expression. The term "tumor associated with AFP expression" generally refers to altered AFP expression in the tumor microenvironment or tumor cells compared to normal cells. For example, the "tumor associated with AFP expression" may be a tumor in which the expression level of AFP is upregulated in the tumor microenvironment or tumor cells compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In an AFP-positive tumor, the protein expression level of AFP in tumor cells or the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than in normal cells.

[0325] For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a liver cancer.

[0326] In another aspect, the present application provides a method for preventing and / or treating a disease or condition, comprising administering the isolated antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, or the pharmaceutical composition to a subject in need thereof.

[0327] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with AFP expression. The term "tumor associated with AFP expression" generally refers to altered AFP expression in the tumor microenvironment or tumor cells compared to normal cells. For example, the "tumor associated with AFP expression" may be a tumor in which the expression level of AFP is upregulated in the tumor microenvironment or tumor cells compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In an AFP-positive tumor, the protein expression level of AFP in tumor cells or the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than in normal cells.

[0328] For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a liver cancer.

[0329] The pharmaceutical compositions and methods described herein can be used in combination with other types of cancer treatments, such as chemotherapy, surgery, radiation, gene therapy, etc. The pharmaceutical compositions and methods described herein can be used to treat other disease conditions that depend on an immune response, such as inflammation, immune disorders, and infectious diseases.

[0330] In the present application, the subject may include a human or a non-human animal. For example, the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice, and rats. The non-human animal may also include any non-human animal species, such as livestock animals, rodents, primates, livestock animals, or poultry. The human may be Caucasian, African, Asian, Semitic, or of other races, or heterozygotes of various races. For example, the human may be an elderly person, an adult, an adolescent, a child, or an infant.

[0331] Effective doses in experimental animals can be used to estimate effective doses in humans. For example, Freireich et al. describe the correlation between animal and human doses (based on milligrams per square meter of body surface area) (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximately determined from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, NY, 537 (1970).

[0332] Without being limited to any theory, the following examples are intended merely to illustrate the antigen-binding proteins, chimeric antigen receptors, production methods, uses, etc. of the present application, and are not intended to limit the scope of the present invention.

[0333] Example Example 1 Construction of an artificial synthetic nanobody (VHH antibody) library and screening of anti-AFP / HLA02 TCR-like antibodies 1.1 Construction of artificial synthetic nanobody (VHH antibody) library A mutation strategy for the CDRs of an artificially synthesized nanobody library was determined by comparing the nucleotide sequences and protein structures of 296 non-repeat nanobodies (VHH antibodies) in the Protein Data Bank (PDB) database. Three CDR3 lengths (14, 17, and 21 amino acids) were identified. The commercially available nanobody Caplacizumab (Publication No. NL300966I2) was selected as a scaffold to synthesize the entire nucleotide sequence of the nanobody Caplacizumab. The entire nucleotide sequence of the artificially synthesized Caplacizumab was then cloned into the HP153 phage vector. Single-stranded DNA from the HP153 phage vector was then extracted. Using the extracted single-stranded DNA as a template, nucleotides in the CDRs of the nanobody Caplacizumab were mutated using the Kunkel mutagenesis method to obtain a double-stranded DNA library of the mutated nanobody. The double-stranded DNA was electroporated into competent E. coli SS320 cells pre-infected with M13KO7. After overnight incubation, the phage supernatant was collected, and the final diversity was 1.44 × 10 10 We constructed an artificial synthetic nanobody library, NanoOri_1.0 (Yuanqi Biotechnology (Shanghai) Co., Ltd.), and screened antibody sequences as a seed bank.

[0334] 1.2 Solution-phase panning of synthetic nanobody libraries TG1 E. coli competent cells were plated onto an antibiotic-free bacterial culture plate. The next day, 15 mL of antibiotic-free 2xYT medium was inoculated with a single TG1 colony and cultured overnight at 37°C. Fresh antibiotic-free 2xYT medium was inoculated into 200 μL of the overnight-cultured TG1 bacterial solution, and the TG1 bacterial solution was cultured at 37°C and 250 rpm until the OD600 reached approximately 0.8. This was then used for further analysis.900 μL of streptavidin-containing magnetic beads were taken and uniformly added to three 1.5 mL EP tubes so that each tube was 300 μL. The tubes were placed on a magnetic rack and washed three times with PBS. Two EP tubes were each added with 30 μg of biotin-labeled human TERT540 polypeptide, which were designated as 540-1 and 540-2. The third tube was added with 30 μg of biotin-labeled human AFP polypeptide, which was designated as hAFP. The three 1.5 mL EP tubes were then stored at room temperature. The tube was incubated at RT for 1 hour, placed on a magnetic rack, washed three times with PBS, and 700 μL of the phage library was added to the 540-1 tube. This was incubated at room temperature for 1 hour, placed on a magnetic rack, and the supernatant was collected. The supernatant was added to the hAFP tube and incubated at room temperature for 1 hour, placed on a magnetic rack, and washed 10 times with PBST (Tween 0.05%). 500 μL of Gly-HCl (pH 2.2) was added to elute the phages. The tube was left to stand at room temperature for 15 minutes, and then placed on a magnetic rack. The tubes were placed on a magnetic rack, and the supernatant was collected. 250 μL of Tris-HCl (pH 8.0) was added to neutralize the supernatant. The neutralized supernatant was added to a 540-2 tube and incubated at room temperature for 1 hour. The tubes were then placed on a magnetic rack, and the supernatant was collected to obtain the secondary library. 20 mL of the previously prepared TG1 bacterial solution (OD600 = 0.8) was added to the secondary library, and the mixture was left to stand at 37°C for 30 minutes. 1 mL of the mixture was then taken and counted. 40 μL of helper phage M13K07 was added, and the mixture was left to stand at 37°C for 30 minutes. The antibiotics ampicillin and kanamycin were added, and 20 mL of 2xYT medium was added and the mixture was cultured overnight at 37°C and 220 rpm. The overnight culture was centrifuged at 8000 rpm and the supernatant was collected. PEG-NaCl precipitate was added in an amount of 1 / 5 the supernatant volume, and the mixture was incubated in an ice bath for 1 hour and centrifuged at 8000 rpm for 30 minutes. The supernatant was removed and the precipitate was resuspended in 1 mL of PBS. This constituted the first round of panning, and the resulting phage was then used for a second round of panning. During each subsequent round of panning, the screening pressure could be adjusted to achieve the desired goal. The specific conditions are shown in Table 1. A total of five rounds of panning were completed.As shown in Table 1, the panning results showed clear enrichment from the third round onwards, and the ELISA results for five rounds of panning are shown in Figure 1. The secondary antibody used was M13 phage antibody (HRP) (manufacturer: Sino Biological).

[0335] [Table 1]

[0336] The monoclonals were selected and subjected to sequencing and identification analysis, and sequence alignment analysis was performed using the software BioEdit. The statistical results are shown in Figure 2. The selected monoclonals included a total of 13 overlapping VHH antibody 1B3 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 1), 11 overlapping VHH antibody 1C4 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 2), 5 overlapping VHH antibody 1C11 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 3), 4 overlapping VHH antibody 1D12 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 4), and 4 overlapping VHH antibody 2F9 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 5), as well as 8 non-overlapping VHH antibody single nucleotide sequences.

[0337] Example 2 Expression and characterization of candidate VHH-Fc antibodies 2.1 Construction of eukaryotic expression vectors for candidate VHH-Fc antibodies and antibody purification Primers were designed and PCR amplification was performed on the candidate VHH antibody nucleotide sequences obtained in Example 1 (the nucleotide sequences are shown in any one of SEQ ID NOs: 1 to 13). Each was then cloned by homologous recombination into an ampicillin-resistant pcDNA3.4 vector that had been double-digested with the restriction endonucleases SfiI and NotI, such that the 3' end of the VHH antibody nucleotide sequence was linked to the 5' end of the antibody Fc portion (SEQ ID NO: 125), thereby constructing a eukaryotic expression vector for the candidate VHH-Fc antibody. The map of the constructed vector is shown in Figure 5. The correctly sequenced recombinant plasmids were transfected into Expi293F cells for transient expression and purified using a Protein A column to obtain different candidate VHH-Fc antibodies. The PCR results are shown in Figure 3, which, from left to right, show a 100-bp marker, 1B3 (VHH antibody 1B3 nucleotide sequence), 1C4 (VHH antibody 1C4 nucleotide sequence), 1C11 (VHH antibody 1C11 nucleotide sequence), 1D12 (VHH antibody 1D12 nucleotide sequence), and 2F9 (VHH antibody 2F9 nucleotide sequence) (approximately 400 bp). An electropherogram of the purified proteins is shown in Figure 4, which, from left to right, shows a marker, reduced 1B3 (VHH-Fc antibody containing the VHH antibody 1B3 nucleotide sequence), and non-reduced 1B3. The molecular weight of purified 1B3 was accurate (approximately 41 kDa after reduction).

[0338] 2.2 Identification of the affinity of candidate VHH-Fc antibodies Using the ForteBio AHC sensor, 100 nM of candidate VHH-Fc antibodies were first bound to the human AFP158-166 / HLA-A02*01* or human TERT540 / HLA-A02*01* complex, respectively. Starting from 150 nM, the antibodies were diluted 2-fold and then bound to the human AFP158-166 / HLA-A02*01* or human TERT540 / HLA-A02*01* complex at seven concentrations. The binding time was 5 minutes and the dissociation time was 10 minutes. Data analysis was performed. The affinity data were analyzed using ELISA kit 9.0. The data results are shown in Table 2, and the affinity results are shown in Figure 6. Among them, two antibodies, No. 17 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 17, the antibody nucleotide sequence is shown in SEQ ID NO: 11) and No. 24 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 24, the antibody nucleotide sequence is shown in SEQ ID NO: 12), showed almost no dissociation, and the affinity of No. 3 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 3, the antibody nucleotide sequence is shown in SEQ ID NO: 6) was higher than that of the Yuruike positive control antibody Ab61 (amino acid sequence SEQ ID NO: 86, nucleotide sequence SEQ ID NO: 85, Fc amino acid sequence SEQ ID NO: 86). The affinity of VHH-Fc antibody No. 4 (a VHH-Fc antibody comprising the nucleotide sequence of VHH antibody No. 4, the antibody nucleotide sequence of which is shown in SEQ ID NO: 7), No. 29 (a VHH-Fc antibody comprising the nucleotide sequence of VHH antibody No. 29, the antibody nucleotide sequence of which is shown in SEQ ID NO: 13), 1B3 (a VHH-Fc antibody comprising the nucleotide sequence of VHH antibody 1B3, the antibody nucleotide sequence of which is shown in SEQ ID NO: 1), and 1C4 (a VHH-Fc antibody comprising the nucleotide sequence of VHH antibody 1C4, the antibody nucleotide sequence of which is shown in SEQ ID NO: 2) was also better than that of the Ulrich positive control antibody Ab61.

[0339] [Table 2]

[0340] 2.3 Identification of non-specific binding activity of candidate VHH-Fc antibodies to different human endogenous polypeptides The antibody binding activity was detected using an iQue Screener flow cytometer. Specifically, the T2 cells were HLA-A02*01* positive cells, and the antibody binding activity was determined to be human AFP. 158 polypeptide (the amino acid sequence of which is shown in SEQ ID NO: 90 and the nucleic acid sequence of which is shown in SEQ ID NO: 89), human NY-Eso-1 157 polypeptide (the amino acid sequence of which is shown in SEQ ID NO: 91) and IFI30 (the amino acid sequence of which is shown in any one of SEQ ID NOs: 92 to 95), BTG2 (the amino acid sequence of which is shown in SEQ ID NO: 96), BCR (the amino acid sequence of which is shown in SEQ ID NO: 97), SSR1 (the amino acid sequence of which is shown in any one of SEQ ID NOs: 98 to 99), PPP2R1B (the amino acid sequence of which is shown in SEQ ID NO: 100), DDX5 (the amino acid sequence of which is shown in SEQ ID NO: 101), CTSG (the amino acid sequence of which is shown in SEQ ID NO: 102), CD247 (the amino acid sequence of which is shown in SEQ ID NO: 103), DMTN (the amino acid sequence of which is shown in SEQ ID NO: 104), CALR (the amino acid sequence of which is shown in SEQ ID NO: 105), Twenty human endogenous polypeptides, including HLA-E (amino acid sequence shown in SEQ ID NO: 105), PIM1 (amino acid sequence shown in SEQ ID NO: 106), HLA-E (amino acid sequence shown in SEQ ID NO: 107), RPS6KB1 (amino acid sequence shown in SEQ ID NO: 108), CSF2RA (amino acid sequence shown in SEQ ID NO: 109), IL7 (amino acid sequence shown in SEQ ID NO: 110), and human TERT540 polypeptide (amino acid sequence shown in SEQ ID NO: 111), were loaded, respectively. These endogenous polypeptides are typically expressed in multiple types of nuclear human cell proteins, such as hemoglobin α chain, β chain, nuclear protein p68, and its analogs. Human AFP158 / T2 was a positive binding cell, and human TERT540 / T2, human NY-Eso-1 157 / T2, and the other 19 human endogenous mixed polypeptides / T2 were non-specific control cells. The cells were incubated in a buffer at a concentration of 1 × 10 6 Prepare cells at 3 x 10 cells / mL and add to a 96-well pointed bottom plate, 30 µL per well to 3 x 10 cells / mL. 4The specific binding activity of candidate VHH antibodies was further identified by dispensing detection antibodies in buffer, starting at 10 μg / mL and then diluting the antibodies 3-fold to set up seven concentration gradients. The results are shown in Figure 7A. 1D12 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1D12), 2F9 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody 2F9), and 1B3 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1B3) showed no nonspecific binding activity to human TERT540 / T2 cells. However, at a high concentration of 10 μg / mL, 1C11 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1C11), 1C4 (a VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1C4), and the Ulrich positive control antibody Ab61 all showed nonspecific binding activity to human TERT540 / T2 cells. As shown in Figure 7B, 1C11, 1C4, 2F9, 1D12, and 1B3 were all able to bind to human AFP158 / T2 cells, and all five candidate VHH-Fc antibodies had no nonspecific binding activity to the human NY-Eso-1 157 / T2 control polypeptide or the human endogenous mixed polypeptide / T2.

[0341] 2.4 Identification of cross-reactivity of candidate VHH-Fc antibodies with murine AFP158 peptide (amino acid sequence shown in SEQ ID NO: 126) Cross-reactivity with the mouse AFP158 peptide / HLA-A*02:01 complex on the surface of live cells was further confirmed. The mouse AFP158 peptide differed from the human AFP158 peptide at two amino acid positions: position 4 and position 9. Antibodies cross-reactive with both the human and mouse AFP158 peptide / MHC complex were used to evaluate antibody drug toxicity in HLA-A02*01* transgenic mice. The results, shown in Figure 7C, showed that all five candidate VHH-Fc antibodies (VHH-Fc antibodies containing the nucleotide sequences of VHH antibodies 1C11, 1C4, 2F9, 1B3, and 1D12) bound to mouse AFP158 / T2 cells, whereas the Yuruike positive control antibody Ab61 showed relatively weak binding activity.

[0342] Example 3 Construction of CAR core plasmids containing candidate VHH antibody nucleotide sequences, lentiviral packaging and production of CAR-T cells 3.1 Construction of CAR core plasmids containing VHH antibody nucleotide sequences The core plasmid was double-digested using the restriction endonucleases SphI and NotI, and then mixed with the candidate VHH antibody nucleotide sequence at a molar ratio of 1:3 to perform homologous recombination. The nucleotide sequence of the homologously recombined core plasmid was sequenced and verified, and the plasmid was amplified and stored for use. The core plasmid map is shown in Figure 8, and the structure of the CAR core plasmid nucleotide sequence is shown in Figure 9. 41BB and CD3ζ were selected as costimulatory domains.

[0343] 3.2. Lentiviral Packaging and Titering The vector system for constructing the lentiviral plasmid of the present invention belongs to a third-generation lentiviral vector system, and the system has a total of three plasmids: (1) packaging plasmid psPAX2 encoding Gag-Pol and Rev proteins; (2) PMD2.G plasmid encoding envelope protein VSV-G; and (3) CAR core plasmid containing the VHH antibody nucleotide sequence constructed according to 3.1. In the core plasmid based on the BBz platform plasmid, the expression of the CAR-encoding gene was regulated by the elongation factor-1α (EF-1α) promoter. The lentiviral packaging process is as follows: 1×10 6293T cells were suspended in 2 mL of DMEM medium containing 10% FBS and seeded into one well of a 6-well plate. After overnight incubation, 144 μL of medium (50 times the mass of the plasmid) was aspirated and mixed with 144 μL of Opti-MEM medium containing 2.88 μg of packaging plasmid (psPAX2:PMD2.G:core plasmid = 3:2:4) and 8.64 μL of FuGENEHD transfection reagent. The mixture was gently mixed uniformly and incubated at 37°C in a CO2 incubator for 12 hours. The medium containing the plasmid was removed, washed once with PBS, and replaced with 2 mL of DMEM medium containing 5% FBS. The medium was then incubated for 48 hours. 2.5 mL of viral supernatant was collected and centrifuged at 3000 rpm for 5 minutes. The supernatant was aliquoted and frozen at -80°C for storage. The titer was then determined for use.

[0344] 3.3 Production of CAR-T cells containing VHH antibodies The method for producing CAR-T cells containing VHH antibodies is as follows: human peripheral blood mononuclear cells were obtained by density gradient centrifugation and grown in a medium containing 200 U / mL interleukin-2 at a cell density of 2 × 10 6 The peripheral blood mononuclear cells were resuspended to a cell density of 0.6 × 10 / mL, and CD3 / CD28 magnetic beads were added at a ratio of 1:3 (cells:magnetic beads) to activate the T cells. The activated peripheral blood mononuclear cells were placed in a CO2 incubator at 37°C and cultured for 24 hours. The lentiviral supernatant obtained above was added at a multiplicity of infection (MOI) of 3, and Polybrene was added to a final concentration of 5 μg / mL. The cell suspension was placed in a well plate and centrifuged at 1200 rpm for 1 hour using a horizontal centrifuge. The well plate was returned to a CO2 incubator at 37°C and cultured for 24 hours. The well plate was centrifuged at 300 g for 5 minutes, the supernatant was removed, and the cells were cultured in fresh X-VIVO medium (manufacturer: LONZA) containing 500 U / mL interleukin-2 at a cell density of 0.6 × 10 6 The cells were resuspended at a cell density of 0.6 × 10 / mL and placed in a CO2 incubator at 37°C. The cells were counted every two days and fresh X-VIVO medium (manufacturer: LONZA) containing 500 U / mL interleukin-2 was added to maintain a cell density of 0.6 × 10 / mL.6 CAR-T cells were cultured for 9-14 days, and the cell positivity was measured. The lentivirus used to infect the cells carried GFP. After the cells were infected with the lentivirus, the GFP positivity was measured using a flow cytometer to obtain the CAR expression positivity rate. Cells with a positivity rate of >20% could be used in tumor killing experiments.

[0345] Example 4 Construction and detection of overexpression cell lines HepG2-MiniG cells and SK-HEP-1-MiniG cells Human AFP was synthesized using lentiviral packaging and lentiviral infection. 158-166The gene (SEQ ID NO: 89) was introduced into HepG2-MiniG and SK-HEP-1 cells, respectively, to produce HepG2-MiniG and SK-HEP-1-MiniG cells. For the construction of SK-HEP-1-MiniG cells, pLV-C-GFPS was used as a vector, and the human AFP158-166 nucleotide sequence was inserted to construct the recombinant plasmid pLV-C-GFPS-AFP-short (SEQ ID NO: 89). The PCR and enzyme digestion results are shown in Figure 10. The PCR amplification results for the human AFP158-166 nucleotide sequence are as follows: (A) From left to right, the 100 bp marker, human AFP158-166 band (approximately 150 bp). The enzyme digestion results for the vector are as follows: (B) Marker, IV. The band (approximately 7500 bp) after vector enzyme digestion. After lentivirus infection, the recombinant plasmid pLV-C-GFPS-AFP-short was transfected into SK-HEP-1 cells. Monoclonal SK-HEP-1-MiniG cells were selected and tested for binding activity by flow cytometry. The specific results are shown in Figure 11. Monoclonal SK-HEP-1-MiniG-7 (SK-HEP-1-MiniG cell monoclonal no. 7) had the highest mean fluorescence intensity and was expanded as the target cell line. HepG2-MiniG cells were constructed using the same method as SK-HEP-1-MiniG cells. The positive rates of HepG2-MiniG cells and SK-HEP-1-MiniG monoclonal cells after expansion were detected by flow cytometry. The results are shown in Figure 12. The positive rates of HepG2-MiniG cells and SK-HEP-1-MiniG cells reached 87.5% and 93.9%, respectively.

[0346] Example 5 In vitro killing and factor detection of CAR-T cells containing candidate VHH antibodies 5.1 In vitro repeated stimulation experiments of CAR-T cells containing candidate VHH antibodies CAR-T cells were produced according to Example 3, and after expansion for 9 to 12 days, the positive rate was detected. CAR-T cells were then extracted and cultured in serum-free X-VIVO medium (manufacturer: LONZA) at a density of 4 × 10 5HepG2-MiniG cells prepared in Example 4 were resuspended in serum-free X-VIVO medium (manufacturer: LONZA) at a density of 4 × 10 5 The effector cells were resuspended at 1 × 10 / mL and used as target cells. The effector cells and target cells were mixed at a 1:1 ratio and cultured in a 37°C, 5% CO2 incubator. The medium color was observed every two days. If the medium color changed from orange to yellow, 1x the volume of medium was added. Cell counting was performed on the fourth or fifth day to determine the amplification factor. After counting, 5 × 10 cells were cultured. 5 The CAR-T cells were further subjected to the second, third, and fourth rounds of amplification in the same manner as above. The amplification fold of the first round was multiplied by the amplification fold of the second, third, and fourth rounds to calculate the total cumulative amplification fold.

[0347] As shown in Figure 13A, after three rounds of repeated stimulation, the cumulative amplification fold of CAR-T cell 1B3 was equivalent to that of CAR-T cell Ab61. As shown in Figure 13B, after four rounds of repeated stimulation, the cumulative amplification fold of CAR-T cells 3, 4, 7, 8, 17, and 29 containing candidate VHH antibodies all exceeded that of CAR-T cell Ab61.

[0348] 5.2 In vitro killing experiments of CAR-T cells containing candidate VHH antibodies The SK-HEP-1-MiniG cells prepared in Example 4 were used as target cells, the CAR-T cells containing the candidate VHH antibodies prepared in Example 3 were used as effector cells, and T cells not infected with lentivirus were used as control effector cells. The specific experimental procedure is as follows: the infection efficiency of the packaged CAR core plasmid lentivirus prepared in Example 3 was detected, and the infection rate of T cells not infected with lentivirus was adjusted so that each group was consistent. The effector cell:target cell (effector cell to target cell ratio) was 1:1, and the target cell number was 1 x 10 4The experimental group consisted of wells containing only effector cells at an equivalent amount to the experimental group, which served as the effector cell self-release background group. The target cell self-release background group consisted of wells containing only target cells at an equivalent amount to the experimental group. The resulting cells were cultured in a CO2 incubator at 37°C for 18 hours. 20 μL of 10x lysis solution was added to the wells containing only a portion of the target cells and incubated for 45 minutes to determine maximum target cell release. The resulting cell culture well plates were centrifuged at 300 g for 5 minutes, and 50 μL of supernatant was collected and used to detect the release of lactate dehydrogenase (LDH). The detection method was described in the CytoTox96 Non-Radioactive Cytotoxicity Reagent Kit (manufacturer: Promega). The released LDH could be detected in the culture supernatant by a coupling enzyme reaction. The formula for calculating cell killing activity is as follows: Killing toxicity %=100×(experimental group−effector cell self-release−target cell self-release+medium background value) / (maximum target cell release−target cell self-release).

[0349] Based on the cell killing formula, the target cell killing activity of CAR-T cells containing each candidate VHH antibody was analyzed, and candidate VHH antibodies with significant killing activity were selected for in vivo functional evaluation.

[0350] The killing results, as shown in Figure 13C, showed that the killing effects of the five candidate VHH antibodies against HLA-A02*01*+ / AFP+ positive SK-HEP-1-MiniG cells were not significantly different. In the cell-killing results using HepG2-MiniG as target cells, CAR-T cells containing candidate VHH antibody 1C11 had a superior target-cell killing effect to CAR-T cells containing the positive control antibody Ab61, but CAR-T cells containing candidate VHH antibody 2F9 had a superior target-cell killing effect to low-expressing HLA-A02*01*+ / AFP+ positive HepG2 cells compared to CAR-T cells containing the positive control antibody Ab61. As shown in Figure 13F, the results showed that the killing activity of CAR-T cells containing candidate VHH antibodies 3, 4, 7, 8, 15, 17, 24, and 29 against HLA-A02*01*+ / AFP+ positive SK-HEP-1-MiniG cells was comparable to that of CAR-T cells containing the positive control antibody Ab61. In cell killing results using HepG2-MiniG as target cells, the cell killing activity of CAR-T cells containing candidate VHH antibodies 3 and 4 was clearly superior to that of CAR-T cells containing the positive control antibody Ab61.

[0351] 5.3 In vitro cytokine secretion experiments of CAR-T cells containing candidate VHH antibodies The HepG2-MiniG or SK-HEP-1-MiniG cells prepared in Example 4 were used as target cells, the CAR-T cells containing the candidate VHH antibodies prepared in Example 3 were used as effector cells, and T cells not infected with lentivirus were used as control effector cells. The specific experimental procedure is as follows: the infection efficiency of the packaged CAR core plasmid lentivirus prepared in Example 3 was detected, and the proportion of T cells not infected with the virus was adjusted so that each group was consistent. The effector cell:target cell (effector cell to target cell ratio) was 1:1, and the target cell number was 2 x 10 4The experimental group consisted of 10 wells containing only effector cells in an amount equal to that of the experimental group, and the background group consisted of wells containing only effector cells in an amount equal to that of the experimental group. The cells were cultured in a CO2 incubator at 37°C for 18 hours, and the resulting cell culture well plates were centrifuged at 300g for 5 minutes to collect 50μL of supernatant, which was used to detect IL2 and IFN-γ expression levels. The detection method was as described in the instruction manual for the R&D DuoSet ELISA Reagent Kit (manufacturer: R&D SYSTEM).

[0352] As shown in Figure 13D, when the target cells were HepG2-MiniG or SK-HEP-1-MiniG, CAR-T cells containing candidate VHH antibodies 1B3, 1C4, and 1C11 secreted high levels of IFN-γ cytokine. As shown in Figure 13E, CAR-T cells containing all candidate VHH antibodies secreted relatively low levels of IL-2 cytokine against HepG2-MiniG cells and non-virus-infected T cells. As shown in Figure 13G, when the target cells were SK-HEP-1-MiniG, CAR-T cells containing candidate VHH antibodies 3, 4, 7, 8, 15, 17, and 1B3 secreted relatively high levels of IFN-γ cytokine. As shown in Figure 13H, when the target cells were HepG2-MiniG cells or SK-HEP-1-MiniG cells, CAR-T cells containing candidate VHH antibodies 15, 17, and 1B3 secreted relatively good IL2 cytokines, and CAR-T cells of all candidate VHH antibodies had relatively low IL2 backgrounds against HepG2-MiniG cells and non-virus-infected T cells.

[0353] Example 6: In vivo efficacy verification of VHH antibody CAR-T cells CAR-T cells containing candidate VHH antibody 17 and CAR-T cells containing candidate VHH antibody 1B3 were randomly selected and subjected to in vivo efficacy evaluation. 6 × 10 cells were injected into 6 mice. 6 Inject K-HEP-1-MiniG cells into the right upper extremity or back subcutaneously, and measure the tumor size to 100 mm. 3When the mice grew to 1000 mm Hg, they were divided into seven groups, each administered by intratumoral or tail vein injection. There were seven groups: one group receiving uninfected T cells (control group IV), two groups receiving CAR-T cells containing the positive control antibody Ab61 (Ab61-IT was the intratumoral injection group, and Ab61-IV was the caudal vein injection group), two groups receiving CAR-T cells containing the candidate VHH antibody 1B3 (1B3-IT was the intratumoral injection group, and 1B3-IV was the caudal vein injection group), and two groups receiving CAR-T cells containing the candidate VHH antibody 17 (17-IT was the intratumoral injection group, and 17-IV was the caudal vein injection group). There were five mice per group, and each group received 1 x 10 CAR-T cells. 7 The CAR-T cells were re-injected every two weeks, and changes in the body weight and tumor size of the mice in each group after administration were recorded to observe their various biological responses.

[0354] In the case of PG(D52), the tumor volume and tumor inhibition rate for each group are shown in Table 3. The mean tumor volume of the non-virus-infected T cell group was 973.98 ± 136.05 mm 3 Compared with the group of T cells not infected with the virus (control group IV), the mean tumor volume of mice in the group intratumorally injected with CAR-T cells containing the positive control antibody Ab61 (Ab61-IT), the group intratumorally injected with CAR-T cells containing the candidate VHH antibody 1B3 (1B3-IT), the group intratumorally injected with CAR-T cells containing the candidate VHH antibody 17 (17-IT), the group via the tail vein injected with CAR-T cells containing the positive control antibody Ab61 (Ab61-IV), the group via the tail vein injected with CAR-T cells containing the candidate VHH antibody 1B3 (1B3-IV), and the group via the tail vein injected with CAR-T cells containing the candidate VHH antibody 17 (17-IV) was 2.73 ± 1.71 mm. 3 , 3.38±2.11mm 3 , 9.58±1.12mm 3 , 19.72±2.08mm 3 , 24.8±1.08mm 3 , 38.32±1.33mm 3Compared with the non-virus-infected T cell group (control group IV), all were significantly different (P<0.0001), with tumor volume inhibition rates of 109.68%, 109.57%, 108.88%, 107.75%, 107.19%, and 105.67%, respectively.

[0355] [Table 3]

[0356] The animal experiment flow is shown in Figure 14. As shown in the tumor growth graph in Figure 15A, the tumors in each group of mice began to shrink continuously 14 days after the first reinfusion of each CAR-T cell. When the average tumor volume of mice in each experimental group was compared with the control group, there was a significant difference (0.0001

Claims

1. The following properties: 1) specifically binds to the human AFP158-166 / HLA-A02*01* complex with a K value of about 3.1E-09M or less; 2) Ability to bind to the mouse AFP158 / / HLA-A02*01* complex; having one or more of: Isolated antigen-binding proteins.

2. 2. The isolated antigen binding protein of claim 1, comprising an HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:

56.

3. 3. The isolated antigen binding protein of claim 1, comprising an HCDR2, wherein the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO:

55.

4. 4. The isolated antigen binding protein of claim 1, comprising HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO:

50.

5. 5. The isolated antigen-binding protein of any one of claims 1 to 4, comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOs: 72 to 84.

6. 6. The isolated antigen binding protein of any one of claims 1 to 5, comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:56; the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO:55; and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:

50.

7. The HCDR1, HCDR2, and HCDR3 are a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19; b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35; e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46; i) HCDR1: SEQ ID NO: 50, HCDR2: SEQ ID NO: 51, and HCDR3: SEQ ID NO: 52; j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56; and k) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; 7. The isolated antigen-binding protein of claim 6, comprising any one of the following amino acid sequences:

8. 8. The isolated antigen binding protein of claim 1, comprising an H-FR1, wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65.

9. 9. The isolated antigen binding protein of claim 1, comprising an H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:

66.

10. 10. The isolated antigen binding protein of any one of claims 1 to 9, comprising an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69.

11. 11. The isolated antigen binding protein of claim 1, comprising an H-FR4, wherein the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

12. 12. The isolated antigen binding protein of any one of claims 1 to 11, comprising H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs:58 to 65, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:66, the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs:67 to 69, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs:70 to 71.

13. The H-FR1, H-FR2, H-FR3, and H-FR4 are a) H-FR1: SEQ ID NO: 58, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, b) H-FR1: SEQ ID NO: 59, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; c) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; d) H-FR1: SEQ ID NO: 61, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; e) H-FR1: SEQ ID NO: 62, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; f) H-FR1: SEQ ID NO: 63, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; g) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, h) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 68, and H-FR4: SEQ ID NO: 71, and i) H-FR1: SEQ ID NO: 65, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 69, and H-FR4: SEQ ID NO: 70; 13. The isolated antigen-binding protein of claim 12, comprising any one of the following amino acid sequences:

14. 14. The isolated antigen-binding protein of any one of claims 1 to 13, comprising a heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 72 to 84.

15. 15. The isolated antigen-binding protein of any one of claims 1 to 14, comprising an antibody or antigen-binding fragment thereof.

16. 16. The isolated antigen-binding protein of claim 15, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb.

17. 17. The isolated antigen-binding protein of any one of claims 1 to 16, comprising a VHH or an antigen-binding fragment thereof.

18. 18. The isolated antigen-binding protein of any one of claims 15 to 17, wherein the antibody is selected from the group consisting of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, and a fully human antibody.

19. 19. The isolated antigen-binding protein of any one of claims 1 to 18, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 72 to 84.

20. A chimeric antigen receptor comprising a targeting portion, wherein the targeting portion comprises an HCDR3, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:

56.

21. 21. The chimeric antigen receptor of claim 20, wherein the targeting portion comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO:

55.

22. The chimeric antigen receptor of any one of claims 20 to 21, wherein the targeting moiety comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, and SEQ ID NO:

50.

23. The chimeric antigen receptor of any one of claims 20 to 22, wherein the targeting portion comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOs: 72 to 84.

24. 24. The chimeric antigen receptor of claim 20, wherein the targeting portion comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:56, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:45, SEQ ID NO:51, and SEQ ID NO:55, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:

50.

25. The HCDR1, HCDR2, and HCDR3 are a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19; b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35; e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25; g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46; i) HCDR1: SEQ ID NO: 50, HCDR2: SEQ ID NO: 51, and HCDR3: SEQ ID NO: 52; j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56; and k) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30; The chimeric antigen receptor of claim 24, comprising any one of the following amino acid sequences:

26. The chimeric antigen receptor of any one of claims 20 to 25, wherein the targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65.

27. The chimeric antigen receptor of any one of claims 20 to 26, wherein the targeting moiety comprises H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence set forth in SEQ ID NO:

66.

28. The chimeric antigen receptor of any one of claims 20 to 27, wherein the targeting moiety comprises H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69.

29. The chimeric antigen receptor of any one of claims 20 to 28, wherein the targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

30. 30. The chimeric antigen receptor of claim 20, wherein the targeting moiety comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 58 to 65, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 66, the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 69, and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 70 to 71.

31. The H-FR1, H-FR2, H-FR3, and H-FR4 are a) H-FR1: SEQ ID NO: 58, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, b) H-FR1: SEQ ID NO: 59, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; c) H-FR1: SEQ ID NO: 60, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; d) H-FR1: SEQ ID NO: 61, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; e) H-FR1: SEQ ID NO: 62, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; f) H-FR1: SEQ ID NO: 63, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70; g) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 67, and H-FR4: SEQ ID NO: 70, h) H-FR1: SEQ ID NO: 64, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 68, and H-FR4: SEQ ID NO: 71, and i) H-FR1: SEQ ID NO: 65, H-FR2: SEQ ID NO: 66, H-FR3: SEQ ID NO: 69, and H-FR4: SEQ ID NO: 70; The chimeric antigen receptor of claim 30, comprising any one of the following amino acid sequences:

32. 32. The chimeric antigen receptor of any one of claims 20 to 31, wherein the targeting moiety comprises an antibody or antigen-binding fragment.

33. The chimeric antigen receptor of claim 32, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

34. The chimeric antigen receptor of any one of claims 20 to 33, wherein the targeting moiety comprises a VHH.

35. The chimeric antigen receptor of claim 34, wherein the VHH targets the human AFP158-166 / HLA-A02*01* complex.

36. The chimeric antigen receptor of any one of claims 20 to 35, wherein the targeting moiety comprises an amino acid sequence set forth in any one of SEQ ID NOs: 72 to 84.

37. The chimeric antigen receptor of any one of claims 20 to 36, comprising a hinge region.

38. 38. The chimeric antigen receptor of claim 37, wherein the hinge region comprises a hinge region derived from a protein selected from the group consisting of IgG4, IgG1, and CD8.

39. The chimeric antigen receptor of any one of claims 37 to 38, wherein the hinge region comprises the amino acid sequence set forth in SEQ ID NO:

147.

40. The chimeric antigen receptor of any one of claims 20 to 39, comprising a transmembrane domain.

41. The chimeric antigen receptor of claim 40, wherein the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, CD24, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD154, and SLAM, or a combination thereof.

42. The chimeric antigen receptor according to any one of claims 40 to 41, wherein the transmembrane domain comprises a transmembrane domain derived from CD8.

43. The chimeric antigen receptor of any one of claims 40 to 42, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

149.

44. The chimeric antigen receptor of any one of claims 40 to 43, wherein the N-terminus of the transmembrane domain is linked to the C-terminus of the hinge region.

45. 45. The chimeric antigen receptor of any one of claims 20 to 44, comprising a costimulatory signaling domain.

46. The chimeric antigen receptor of claim 45, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from a protein 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, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88, or a combination thereof.

47. The chimeric antigen receptor of any one of claims 45 to 46, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from 4-1BB.

48. The chimeric antigen receptor of any one of claims 45 to 47, wherein the costimulatory signaling domain comprises a costimulatory signaling domain derived from CD28.

49. 49. The chimeric antigen receptor of any one of claims 45 to 48, wherein the costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 132 or SEQ ID NO:

130.

50. 50. The chimeric antigen receptor of any one of claims 45 to 49, wherein the N-terminus of the costimulatory signaling domain is linked to the C-terminus of the transmembrane domain.

51. The chimeric antigen receptor of any one of claims 20 to 50, comprising an intracellular signaling domain.

52. The chimeric antigen receptor of claim 51, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, and DAP-12, or a combination thereof.

53. The chimeric antigen receptor according to any one of claims 51 to 52, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from CD3ζ.

54. The chimeric antigen receptor of any one of claims 51 to 53, wherein the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

134.

55. 55. The chimeric antigen receptor of any one of claims 51 to 54, wherein the N-terminus of the intracellular signaling domain is linked to the C-terminus of the costimulatory signaling domain.

56. The chimeric antigen receptor of any one of claims 20 to 55, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 112 to 124.

57. 57. A polypeptide comprising the isolated antigen binding protein of any one of claims 1 to 19 and / or the chimeric antigen receptor of any one of claims 20 to 56.

58. 20. An immunoconjugate comprising the isolated antigen-binding protein of any one of claims 1 to 19.

59. 58. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1 to 19, the chimeric antigen receptor of any one of claims 20 to 56, or the polypeptide of claim 57.

60. 60. A vector comprising the isolated nucleic acid molecule of claim 59.

61. 61. A modified cell comprising the isolated antigen binding protein of any one of claims 1 to 19, the chimeric antigen receptor of any one of claims 20 to 56, the polypeptide of claim 57, the immunoconjugate of claim 58, the isolated nucleic acid molecule of claim 59 and / or the vector of claim 60.

62. 62. The modified cell of claim 61, wherein the modification comprises upregulating the expression level of a low density lipoprotein receptor-related protein or a fragment thereof in the cell.

63. The modified cell of claim 62, wherein the low-density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

64. 64. The modified cell of any one of claims 62 to 63, wherein the low density lipoprotein receptor-related protein or fragment thereof is of human origin.

65. The modified cell of any one of claims 63 to 64, wherein the functional fragment comprises a fragment or truncated body of the low density lipoprotein receptor-related protein having low density lipoprotein receptor-related protein activity.

66. The modified cell of any one of claims 62 to 65, wherein the low-density lipoprotein receptor-related protein comprises low-density lipoprotein receptor-related protein 6 and truncations thereof, and / or low-density lipoprotein receptor-related protein 5 and truncations thereof.

67. A modified cell described in any one of claims 62 to 66, wherein the truncated form of low-density lipoprotein receptor-related protein 6 comprises the intracellular region of low-density lipoprotein receptor-related protein 6 and / or the truncated form of low-density lipoprotein receptor-related protein 5 comprises the intracellular region of low-density lipoprotein receptor-related protein 5.

68. A modified cell described in any one of claims 62 to 67, wherein the truncated form of low-density lipoprotein receptor-related protein 6 comprises the transmembrane region of low-density lipoprotein receptor-related protein 6 and the LDLR region of low-density lipoprotein receptor-related protein 6, and / or the truncated form of low-density lipoprotein receptor-related protein 5 comprises the transmembrane region of low-density lipoprotein receptor-related protein 5 and the LDLR region of low-density lipoprotein receptor-related protein 5.

69. The modified cell of any one of claims 62 to 68, wherein the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, and SEQ ID NO:

144.

70. 70. The modified cell of any one of claims 61 to 69, wherein the modification comprises the introduction of a vector into the modified cell that leads to upregulation of the expression level of the low density lipoprotein receptor-related protein or a fragment thereof.

71. 71. The modified cell of any one of claims 61 to 70, comprising an immune cell.

72. 72. The modified cell of claim 71, wherein the immune cells are selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

73. 73. The modified cell of any one of claims 61 to 72, wherein the cell comprises a T cell.

74. 74. A method of producing an isolated antigen binding protein according to any one of claims 1 to 19, a chimeric antigen receptor according to any one of claims 20 to 56, and / or a polypeptide according to claim 57, the method comprising culturing a modified cell according to any one of claims 61 to 73 under conditions which allow expression of said isolated antigen binding protein and / or said polypeptide.

75. 74. A pharmaceutical composition comprising an isolated antigen binding protein of any one of claims 1 to 19, a chimeric antigen receptor of any one of claims 20 to 56, a polypeptide of claim 57, an immunoconjugate of claim 58, an isolated nucleic acid molecule of claim 59, a vector of claim 60, a modified cell of any one of claims 61 to 73, and / or a pharmaceutically acceptable adjuvant and / or excipient.

76. 59. A method for detecting AFP protein, comprising administering an isolated antigen-binding protein of any one of claims 1 to 19, a polypeptide of claim 57, or an immune complex of claim 58.

77. 59. A kit for detecting AFP protein, comprising the isolated antigen-binding protein of any one of claims 1 to 19, the polypeptide of claim 57, or the immune complex of claim 58.

78. 59. Use of an isolated antigen-binding protein according to any one of claims 1 to 19, a polypeptide according to claim 57, or an immune complex according to claim 58 in the manufacture of a kit for detecting the presence and / or content of AFP protein.

79. 74. Use of an isolated antigen-binding protein according to any one of claims 1 to 19, a chimeric antigen receptor according to any one of claims 20 to 56, a polypeptide according to claim 57, an immunoconjugate according to claim 58, an isolated nucleic acid molecule according to claim 59, a vector according to claim 60, and / or a modified cell according to any one of claims 61 to 73 in the manufacture of a medicament for the prevention and / or treatment of tumors.

80. 80. The use of claim 79, wherein the tumor comprises a solid tumor.

81. 81. The use according to any one of claims 79 to 80, wherein the tumor comprises a non-solid tumor.

82. 82. The use of any one of claims 79 to 81, wherein the tumor comprises a tumor associated with expression of AFP.

83. 83. The use according to any one of claims 79 to 82, wherein the tumor comprises liver cancer.

84. 74. Use of the isolated antigen binding protein of any one of claims 1 to 19, the chimeric antigen receptor of any one of claims 20 to 56, the polypeptide of claim 57, the immunoconjugate of claim 58, the isolated nucleic acid molecule of claim 59, the vector of claim 60, and / or the modified cell of any one of claims 61 to 73 for the prevention and / or treatment of tumors.

85. 85. The use of claim 84, wherein the tumor comprises a solid tumor.

86. 86. The use according to any one of claims 84 to 85, wherein the tumor comprises a non-solid tumor.

87. 87. The use of any one of claims 84 to 86, wherein the tumor comprises a tumor associated with expression of AFP.

88. 88. The use according to any one of claims 84 to 87, wherein the tumor comprises liver cancer.

89. 74. A method for preventing and / or treating a disease or condition, the method comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein of any one of claims 1 to 19, the chimeric antigen receptor of any one of claims 20 to 56, the polypeptide of claim 57, the immunoconjugate of claim 58, the isolated nucleic acid molecule of claim 59, the vector of claim 60, and / or the modified cell of any one of claims 61 to 73.

90. 90. The method of claim 89, wherein the tumor comprises a solid tumor.

91. The method of claims 89-90, wherein the tumor comprises a non-solid tumor.

92. 92. The method of any one of claims 89 to 91, wherein the tumor comprises a tumor associated with expression of AFP.

93. 93. The method of any one of claims 89 to 92, wherein the tumor comprises liver cancer.

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