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

Abstract

The invention relates to an anti-AFP / HLA02TCR-like antibody and application thereof. The antibody can be specifically combined with a human AFP 158-166 / HLA-A02 * 01 * compound at a KD (Ketene Dimer) value of about 3.1 E-09M or below. The invention also relates to a chimeric antigen receptor containing the antibody, an immunoconjugate and a cell containing the chimeric antigen receptor.

Description

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

[0001] The present application relates to the field of biomedicine, and specifically to an anti-AFP / HLA02 TCR-like antibody and its use. Background Art

[0002] Alpha-fetoprotein (AFP) is a glycoprotein. In adults, AFP is elevated in the serum of approximately 80% of liver cancer patients. AFP positivity is found in 50% of germ cell tumors. It can also be elevated to varying degrees in patients with pancreatic cancer, lung cancer, and cirrhosis. Therefore, AFP holds promise as a therapeutic target for a variety of solid tumors.

[0003] Intracellular tumor-specific antigens can be processed into peptides and presented to the surface of tumor cells through class I major histocompatibility complex (MHC). TCR-like antibodies can bind to the peptide / MHC complex, thereby inducing tumor cell death. TCR-like antibodies can be converted into CAR structures to mediate specific tumor lysis through T cells.

[0004] While CART technology has achieved significant success in hematologic malignancies, its application in solid tumors has yet to see significant changes. CART therapy faces several challenges: 1) Unlike leukemia, which spreads throughout the body, solid tumors do not develop. CART cells must reach the lesions and infiltrate the tumor to be effective, a steric barrier to CART therapy created by the solid tumor tissue structure. 2) The inhibitory effects of the tumor's internal immune microenvironment can prevent CART cells from functioning properly and lead to T cell exhaustion. 3) Hypoxia and nutrient deficiencies in the microenvironment of solid tumors are also detrimental to the proliferation of CART cells and the production of targeted cytotoxicity.

[0005] Therefore, there is an urgent need to develop new antibodies and CAR structures to treat solid tumors.

[0006] Summary of the Invention

[0007] The present application provides an isolated antigen-binding protein having one or more of the following properties: 1) a K of about 3.1E-09 M or less; D Value and People AFP 158-166 / HLA-A02*01* complex specifically; and 2) can bind to mouse AFP 158 / / HLA-A02*01* complex binding.

[0008] In one aspect, the present application provides an isolated antigen-binding protein comprising a HCDR3 comprising the amino acid sequence shown 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.

[0009] In certain embodiments, the antigen binding protein comprises a HCDR2 comprising the amino acid sequence of 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.

[0010] In certain embodiments, the antigen binding protein comprises HCDR1, which comprises the amino acid sequence shown 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.

[0011] In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in any one of SEQ ID NO: 72 to SEQ ID NO: 84.

[0012] In certain embodiments, the antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown 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 shown 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 shown 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.

[0013] In certain embodiments, the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one of the following groups:

[0014] a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19;

[0015] b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0016] c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30;

[0017] d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35;

[0018] e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0019] f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0020] g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30;

[0021] h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46;

[0022] i) HCDR1: SEQ ID NO:50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO:52;

[0023] j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56;

[0024] and

[0025] k) HCDR1: SEQ ID NO:37, HCDR2: SEQ ID NO:24, and HCDR3: SEQ ID NO:30.

[0026] In certain embodiments, the antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 58 to SEQ ID NO: 65.

[0027] In certain embodiments, the antigen binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:66.

[0028] In certain embodiments, the antigen binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 67 to SEQ ID NO: 69.

[0029] In certain embodiments, the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0030] In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:65; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:66; the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0031] In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 in the antigen binding protein comprise any one group of amino acid sequences selected from the following:

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] 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

[0040] 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.

[0041] In certain embodiments, the antigen binding protein comprises a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 72 to SEQ ID NO: 84.

[0042] In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0043] In certain 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.

[0044] In certain embodiments, the antigen binding protein comprises a VHH or an antigen binding fragment thereof.

[0045] In certain 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.

[0046] In certain embodiments, the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:72 to SEQ ID NO:84.

[0047] On the other hand, the present application provides a chimeric antigen receptor, which comprises a targeting portion, the targeting portion comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown 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.

[0048] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown 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.

[0049] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown 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.

[0050] In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in any one of SEQ ID NO: 72 to SEQ ID NO: 84.

[0051] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown 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 shown 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 shown 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.

[0052] In certain embodiments, the HCDR1, HCDR2, and HCDR3 of the chimeric antigen receptor comprise an amino acid sequence selected from any one of the following groups:

[0053] a) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, and HCDR3: SEQ ID NO: 19;

[0054] b) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0055] c) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30;

[0056] d) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35;

[0057] e) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0058] f) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 25;

[0059] g) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 30;

[0060] h) HCDR1: SEQ ID NO: 44, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 46;

[0061] i) HCDR1: SEQ ID NO:50, HCDR2: SEQ ID NO:51, and HCDR3: SEQ ID NO:52;

[0062] j) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 55, and HCDR3: SEQ ID NO: 56;

[0063] and

[0064] k) HCDR1: SEQ ID NO:37, HCDR2: SEQ ID NO:24, and HCDR3: SEQ ID NO:30.

[0065] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:65.

[0066] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:66.

[0067] In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69.

[0068] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0069] In certain 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 shown in any one of SEQ ID NO:58 to SEQ ID NO:65; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:66; the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0070] In certain embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 in the chimeric antigen receptor comprise any one group of amino acid sequences selected from the following:

[0071] 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;

[0072] 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;

[0073] 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;

[0074] 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;

[0075] 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;

[0076] 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;

[0077] 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;

[0078] 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

[0079] 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.

[0080] In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises an antibody or an antigen-binding fragment.

[0081] In certain embodiments, the antigen-binding fragment of 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.

[0082] In certain embodiments, the targeting moiety in the chimeric antigen receptor comprises a VHH.

[0083] In certain embodiments, the VHH in the chimeric antigen receptor targets human AFP 158-166 / HLA-A02*01* complex.

[0084] In certain embodiments, the targeting portion of the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:72 to SEQ ID NO:84.

[0085] In certain embodiments, the chimeric antigen receptor comprises a hinge region.

[0086] In certain embodiments, the hinge region of the chimeric antigen receptor comprises a hinge region derived from the following proteins: IgG4, IgG1, and CD8.

[0087] In certain embodiments, the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:147.

[0088] In certain embodiments, the chimeric antigen receptor comprises a transmembrane domain.

[0089] In certain 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.

[0090] In certain embodiments, the transmembrane domain of the chimeric antigen receptor comprises a transmembrane domain derived from CD8.

[0091] In certain embodiments, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:149.

[0092] In certain embodiments, the N-terminus of the transmembrane domain in the chimeric antigen receptor is connected to the C-terminus of the hinge region.

[0093] In certain embodiments, the chimeric antigen receptor comprises a costimulatory signaling domain.

[0094] In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor 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 of CD83, CD40, and MyD88, or a combination thereof.

[0095] In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from 4-1BB.

[0096] In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises a costimulatory signaling domain derived from CD28.

[0097] In certain embodiments, the costimulatory signaling domain in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 132 or SEQ ID NO: 130.

[0098] In certain embodiments, the N-terminus of the costimulatory signaling domain in the chimeric antigen receptor is linked to the C-terminus of the transmembrane domain.

[0099] In certain embodiments, the chimeric antigen receptor comprises an intracellular signaling domain.

[0100] In certain 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, and DAP-12, or a combination thereof.

[0101] In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises an intracellular signaling domain derived from CD3ζ.

[0102] In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:134.

[0103] In certain 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.

[0104] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:112 to SEQ ID NO:124.

[0105] In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein and / or the chimeric antigen receptor.

[0106] On the other hand, the present application provides an immunoconjugate comprising the isolated antigen-binding protein.

[0107] 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.

[0108] In another aspect, the present application provides one or more vectors comprising the isolated nucleic acid molecule.

[0109] 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.

[0110] In certain embodiments, the modification comprises upregulating the expression of low-density lipoprotein receptor-related protein or a fragment thereof in the cell.

[0111] In certain embodiments, the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0112] In certain embodiments, the low-density lipoprotein receptor-related protein or fragment thereof is of human origin.

[0113] In certain embodiments, the functional fragment comprises a fragment or truncation of the low-density lipoprotein receptor-related protein that has the activity of the low-density lipoprotein receptor-related protein.

[0114] In certain embodiments, the low-density lipoprotein receptor-related protein comprises low-density lipoprotein receptor-related protein 6 and its truncations, and / or low-density lipoprotein receptor-related protein 5 and its truncations.

[0115] In certain 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.

[0116] In certain embodiments, the truncation 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 truncation 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.

[0117] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142 and SEQ ID NO: 144.

[0118] In certain embodiments, the modification comprises introducing into the modified cell a vector that upregulates the expression of the low-density lipoprotein receptor-related protein or a fragment thereof.

[0119] In certain embodiments, the modified cells comprise immune cells.

[0120] In certain 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.

[0121] In certain embodiments, the modified cells comprise T cells.

[0122] On the other hand, the present application provides a method for preparing the isolated antigen-binding protein, the chimeric antigen receptor, and / or the polypeptide, which comprises culturing the modified cell under conditions such that the isolated antigen-binding protein and / or the polypeptide are expressed.

[0123] On the other hand, 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.

[0124] In another aspect, the present application provides a method for detecting AFP protein, comprising:

[0125] Administering the isolated antigen binding protein, the polypeptide or the immunoconjugate.

[0126] On the other hand, the present application provides a detection kit for AFP protein, which comprises the isolated antigen-binding protein, the polypeptide or the immunoconjugate.

[0127] On the other hand, the present application provides use of the isolated antigen-binding protein, the polypeptide or the immunoconjugate in preparing a kit for detecting the presence and / or content of AFP protein.

[0128] On the other hand, 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 in the preparation of a drug for preventing and / or treating tumors.

[0129] In certain embodiments, the tumor in the use comprises a solid tumor.

[0130] In certain embodiments, the tumor in the use comprises a non-solid tumor.

[0131] In certain embodiments, the tumor in the use includes a tumor associated with the expression of AFP.

[0132] In certain embodiments, the tumor in the use comprises liver cancer.

[0133] On the other hand, the present application provides 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, which are used for preventing and / or treating tumors.

[0134] In certain embodiments, the tumor in the use comprises a solid tumor.

[0135] In certain embodiments, the tumor in the use comprises a non-solid tumor.

[0136] In certain embodiments, the tumor in the use includes a tumor associated with the expression of AFP.

[0137] In certain embodiments, the tumor in the use comprises liver cancer.

[0138] On the other hand, the present application provides a method for preventing and / or treating a disease or condition, which comprises administering 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 to a subject in need thereof.

[0139] In certain embodiments, the methods described herein comprise a solid tumor.

[0140] In certain embodiments, the methods described herein comprise a non-solid tumor.

[0141] In certain embodiments, the method further comprises a method wherein the tumor comprises a tumor associated with the expression of AFP.

[0142] In certain embodiments, the methods described herein comprise liver cancer.

[0143] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0145] FIG1 shows the results of five rounds of panning ELISA assays described in this application.

[0146] FIG2 shows the sequence reproducibility of the single clones picked after panning described in the present application.

[0147] FIG3 shows the results of PCR amplification of the nucleotide sequences of the candidate VHH antibodies described in this application.

[0148] FIG4 shows the SDS-PAGE results of the expressed and purified VHH antibodies described in the present application.

[0149] FIG5 shows the eukaryotic expression vector of the candidate VHH-Fc antibody described in this application (taking 1C11 as an example).

[0150] FIG6 shows the affinity of the candidate VHH-Fc antibodies described in this application.

[0151] FIG7A shows the flow cytometry results of the candidate VHH-Fc antibodies described in this application and human TERT540 / T2 cells.

[0152] FIG7B shows the flow cytometry results of the candidate VHH-Fc antibodies and polypeptides / T2 cells described in the present application.

[0153] FIG7C shows the flow cytometry results of the candidate VHH-Fc antibodies described in the present application and mouse AFP158 / T2 cells.

[0154] FIG8 shows a map of the core plasmid vector described in this application.

[0155] Figure 9 shows the structure diagram of the CAR core plasmid described in this application.

[0156] FIG10A shows the PCR results of amplifying the nucleotide sequence of human AFP158-166 described in the present application.

[0157] FIG10B shows the result of enzyme digestion of the vector described in this application.

[0158] FIG11 shows the results of flow cytometry screening and detection of monoclonal clones described in the present application.

[0159] FIG12 shows the results of the flow cytometry screening of the positive rates of HEPG2-MiniG and SK-HEP-1-MiniG described in the present application.

[0160] Figures 13A-13B show the results of the CAR-T cell repeated stimulation and expansion experiment described in this application.

[0161] Figures 13C and 13F show the results of an in vitro cell killing experiment with CAR-T cells at an effector-target ratio of 1:1 as described in the present application.

[0162] FIG13D and FIG13G show the results of IFN-γ cytokine secretion at an effector-target ratio of 1:1 as described in the present application.

[0163] FIG13E and FIG13H show the IL-2 cytokine secretion results at an effector-target ratio of 1:1 as described in the present application.

[0164] FIG14 shows a flowchart of the animal experiment described in this application.

[0165] FIG15A shows a tumor growth curve as described in the present application.

[0166] FIG. 15B shows a graph of the animal body weight curve described in the present application. DETAILED DESCRIPTION

[0167] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0168] Definition of terms

[0169] In this application, the term "isolated antigen-binding protein" generally refers to a polypeptide polymer that can specifically recognize and / or neutralize a specific antigen. For example, an isolated antigen-binding protein can include a portion of a heavy chain. For example, an isolated antigen-binding protein can include a heavy chain variable region. The term "isolated antigen-binding protein" can include single-domain antibodies. For example, isolated antigen-binding proteins can include, but are not limited to, human single-domain antibodies.

[0170] In this application, the term "single domain antibody" or "sdAb" or "VHH" generally refers to a class of antibodies that lack an antibody light chain and have only a heavy chain variable region. In some cases, single domain antibodies can be derived from Bactrian camels, dromedaries, alpacas, llamas, nurse sharks, whitebait sharks, or rays (for example, see Kang Xiaozhen et al., Journal of Biotechnology, 2018, 34(12): 1974-1984). For example, single domain antibodies can be derived from alpacas. Single domain antibodies 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 can be further divided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: 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).

[0171] 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 TCR in terms of target recognition. Technically, TCR-like antibodies can be produced by conventional hybridoma technology or by in vitro antibody library technology 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 158- 166 / HLA-A02*01* complex.

[0172] In this application, the term "transmembrane domain" generally refers to a sequence in a cell surface protein that spans the cell membrane and may include a hydrophobic alpha helix. The transmembrane domain may be connected to an intracellular signaling domain, thereby transmitting signals. In this application, the transmembrane domain may be derived from any type I, type II, or type III transmembrane protein. In the present application, membrane-spanning domain can include the membrane-spanning domain or its combination derived from lower histone: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.For example, membrane-spanning domain can include the membrane-spanning domain derived from described CD8.

[0173] In this application, the term "Chimeric Antigen Receptor (CAR)" generally refers to a fusion protein comprising a targeting portion capable of binding to an antigen and at least one intracellular domain. CAR is a core component of chimeric antigen receptor T cells (CAR-T), which may include a targeting portion (e.g., targeting tumor-specific antigens and / or tumor-associated antigens), a signal peptide, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. In this application, the CAR may be based on an antigen of an antibody (e.g., AFP 158-166The CAR is a family of proteins that bind to the HLA-A02*01* complex and are specifically linked to the T cell receptor activation intracellular domain. Genetically modified T cells expressing the CAR can specifically recognize and eliminate malignant cells expressing the target antigen. For descriptions of CAR and CAR-T cells, see, for example, Sadelain M, Brentjens R, Riviere 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.

[0174] In the present application, the term "costimulatory signaling domain" generally refers to an intracellular domain that can provide an immune co-stimulatory molecule, which is a cell surface molecule required for the effective response of lymphocytes to antigens. In some cases, the co-stimulatory signaling domain can include a co-stimulatory signaling domain or a combination thereof 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 of CD83, CD40 and MyD88. For example, the costimulatory signaling domain can include a costimulatory signaling domain derived from 4-1BB.

[0175] In this application, the term "intracellular signaling domain" generally refers to a domain located inside a cell that can transduce a signal. In this application, the intracellular signaling domain can conduct a signal into the cell. Generally, an intracellular signaling domain is any continuous amino acid sequence used to guide protein targeting. In some cases, the intracellular signaling domain may comprise an intracellular signaling domain or a combination thereof 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from CD3ζ.

[0176] In this application, the term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising its antigen-binding portion. 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 antibody fragments that bind to antigens (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, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives 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 can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: 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 region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0177] In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. 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 usually monovalent fragments consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragments, which may be bivalent fragments comprising two Fab fragments linked by a disulfide bond 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, which may be optionally linked by a linker. In addition, monovalent single-chain molecules Fv (scFv) formed by pairing VL and VH may also be included (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883). The "antigen-binding fragment" may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. For example, the antigen-binding fragment may also include a single domain antibody.

[0178] In this application, the term "monoclonal antibody" generally refers to a group of substantially homologous antibodies, i.e., the individual antibodies comprising the group are identical except for possible naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. For example, the monoclonal antibodies can be prepared by hybridoma technology or produced in bacteria, eukaryotic animals, or plant cells using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries 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).

[0179] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequences in another species. For example, the variable regions of the light and heavy chains are both derived from the variable regions of an antibody from one animal species (such as a mouse, rat, etc.), while the constant portion is homologous to the antibody sequence from another species (such as a human). For example, to obtain a chimeric antibody, a non-human B cell or hybridoma cell can be used to produce the variable region, while the constant region combined with it is from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined with it. At the same time, since the constant region of a chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody using a non-human constant region.

[0180] In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while retaining the antibody's full antigen binding affinity and specificity. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof can be used; including "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), "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 The binding domain of non-human origin is humanized using 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), surface reconstruction (U.S. Pat. No. 5,639,641). If other regions, such as hinge regions and constant region domains, are also derived from non-human sources, these regions can also be humanized.

[0181] In this application, the term "ribosome skip site," also known as an internal ribosome entry site (IRES), generally refers to a nucleotide sequence located in the middle of an mRNA sequence that is reserved for translation initiation. The ribosome skip site can allow translation to be initiated in a cap-independent manner. The IRES is typically located in the 5' UTR. In this application, the ribosome skip site may include sequences from positions 1 to 578 of SEQ ID NO: 145.

[0182] In the present application, 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 the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in the tumor compared with normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression of AFP in the tumor microenvironment or in the tumor is upregulated compared with normal cells. The tumor associated with the protein expression of AFP may be an AFP-positive tumor. In an AFP-positive tumor, the protein expression of AFP in tumor cells or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that in normal cells.

[0183] In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other therapeutic agent to the isolated antigen-binding protein (e.g., covalently linked via a linker molecule), which can deliver the other therapeutic agent to the target cell (e.g., tumor cell) through the specific binding of the isolated antigen-binding protein to the antigen on the target cell. In addition, the antigen can also be secreted by the target cell and located in the space outside the target cell.

[0184] In this application, the term "K D ”(Similarly, “K D ” or “K D ”) is often referred to as the “affinity constant” or “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (kd) by the association rate constant (ka). The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K) are used to D ) represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein as described herein) to an antigen (e.g., an AFP protein). Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be measured by Biacore (biomolecular interaction analysis) (e.g., an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). DThe K value can also be determined using other experimental approaches and instruments such as Octet detection. In addition, the K can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho). D The K value can be measured by using a surface plasmon resonance (SPR) D value.

[0185] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0186] In this application, the term "vector" generally refers to a nucleic acid molecule that can replicate itself in a suitable host. The vector can transfer the inserted nucleic acid molecule into a cell and / or between cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector can produce a desired expression product by culturing suitable cells containing the vector. In this application, the vector may include a lentiviral vector.

[0187] In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing a chimeric antigen receptor described herein or an antigen-binding protein described herein. The cell may include the progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but may be capable of expressing the chimeric antigen receptor or antigen-binding protein described herein. The cell can be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). In some embodiments, the cell may be an immune cell. For example, the immune cell can 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 cell can be a T cell.

[0188] As used herein, the term "treat," ...

[0189] In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications can include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation to a labeling component). The term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and the D and L optical isomers, as well as amino acid analogs and peptide mimetics.

[0190] In this application, 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. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (a locus) defined by linkage analysis, 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. A polynucleotide can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling moiety.

[0191] In addition to the specific proteins and nucleotides mentioned herein, the present application also includes functional variants, derivatives, analogs, homologues and fragments thereof.

[0192] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a naturally occurring sequence or encoded by a nucleotide sequence substantially identical to that of a naturally occurring sequence and capable of possessing 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 particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one amino acid residue and / or nucleotide residue present in a naturally occurring protein and / or polynucleotide, as long as the original functional activity is retained.

[0193] In the present application, the term "derivative" generally refers to any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues in the polypeptide or polynucleotide of the present application, as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0194] In this application, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie, a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.

[0195] Generally, amino acid substitutions can be made, such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

[0196] The proteins or polypeptides used in the present application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing non-charged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0197] In the present application, the term "AFP antigen" generally refers to a glycoprotein that belongs to the albumin family. The amino acid sequence of human AFP can be found in UniProt / Swiss-Prot accession number P02771.

[0198] In this application, the term "T cell", also known as T lymphocyte, is a subtype of white blood cells 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 the T cell receptors present on the cell surface. In this application, the T cells may include memory stem cell-like T cells (TSCM) and central memory T cells (TCM).

[0199] In this application, the term "T cell receptor," often also referred to as "TCR," generally refers to the molecular structure that allows T cells to specifically recognize and bind to antigenic peptides and MHC molecules. The T cell receptor can exist on the surface of T cells in the form of a complex with the CD3 molecule. The TCR can be a heterodimer anchored to the cell membrane, mostly composed of highly variable α and β subunits linked by disulfide bonds; a minority consists of γ and δ peptide chains. The TCR can include a variable region and a constant region, wherein the constant region can be located near the cell membrane, connecting the transmembrane region and the intracellular end, while the variable region is responsible for recognizing the peptide / MHC complex.

[0200] In the present application, the term "low-density lipoprotein receptor-related protein" (Low density lipoprotein receptor-related protein, LRP) generally refers to a mosaic protein comprising 839 amino acids (after removing the 21 amino acid signal peptide). It is embedded in the outer phospholipid layer of LDL (Low density lipoprotein) particles and is an endocytic receptor that can mediate the endocytosis of cholesterol-rich LDL. It is a member of the low-density lipoprotein receptor (LDLR) gene family. The LRP is most significantly expressed in bronchial epithelial cells and adrenal and cortical tissues. In the present application, the low-density lipoprotein receptor-related protein may include one or more selected from the following group: low-density lipoprotein receptor-related proteins 1-12 or truncated forms.

[0201] 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 a unique subgroup of the low-density lipoprotein receptor (LDLR) family. Human LRP-6 has the accession number O75581 in UniProt. Human LRP-5 has the accession number O75197 in UniProt.

[0202] In this application, the term "truncated protein" generally refers to a truncated protein. Such truncations can be obtained by proteolysis or manipulation of the structural gene to eliminate the N- or C-terminal portion of the protein. Alternatively, such truncations can be obtained by nonsense mutations that result in the presence of stop codons in the structural gene, thereby prematurely terminating translation.

[0203] In this application, the term "and / or" should be understood to mean either one of the alternatives or both of the alternatives.

[0204] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0205] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0206] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0207] Detailed Description of the Invention

[0208] The isolated antigen-binding protein described in this application

[0209] In one aspect, the present application provides an isolated antigen binding protein that can be detected with a K of 3.1E-09M or less in a ForteBio AHC sensor. D Value (for example, the K D No more than about 3.1E-09M, no more than about 3.0E-09M, no more than about 2.9E-09M, no more than about 2.8E-09M, no more than about 2.7E-09M, no more than about 2.6E-09M, no more than about 2.7E-09M, no more than about 2.6E-09M, no more than about 2.3E-09M, no more than about 2.0E-09M, no more than about 1.5E-09M, no more than about 1E-09M or no more than 5E-10M or less) and human AFP 158-166 / HLA-A02*01* complex specifically binds.

[0210] In one aspect, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 72 to SEQ ID NO: 84.

[0211] In the present application, the HCDR of the isolated antigen-binding protein can be divided in any form. As long as the VH is identical to the amino acid sequence shown in SEQ ID NO: 72 to SEQ ID NO: 84, the HCDR obtained by any division can fall within the protection scope of the present application.

[0212] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, North's, and Kabat / Chothia combined. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (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) sequence identity with the amino acid sequence of the CDR.

[0213] 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.

[0214] In the present application, the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown 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 HCDR3 sequence of the antigen-binding protein may be defined according to the IMGT numbering system.

[0215] In the present application, the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown 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. For example, the HCDR3 sequence of the antigen-binding protein may be defined according to the Kabat numbering system.

[0216] In the present application, the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the antigen-binding protein may be defined according to the IMGT numbering system.

[0217] In the present application, the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the antigen-binding protein may be defined according to the Kabat numbering system.

[0218] In the present application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown 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. For example, the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT numbering system.

[0219] In the present application, the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown 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 sequence of the antigen-binding protein may be defined according to the Kabat numbering system.

[0220] For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence of 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 of 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 of 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.

[0221] In the present application, the antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown 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 shown 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 shown 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 according to the Kabat numbering system.

[0222] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0223] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0224] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0225] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0226] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0227] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0228] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0229] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0230] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0231] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0232] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0233] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0234] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0235] For example, the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.

[0236] In the present application, the 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.

[0237] In the present application, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:66.

[0238] In the present application, the 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.

[0239] In the present application, the 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.

[0240] In the present application, the H-FR1 of the antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:65; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:66; the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0241] In the present application, H-FR1 of the antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 58; 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 1B3 or an antigen-binding fragment thereof having the same H-FR1-4.

[0242] 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.

[0243] 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 sequences.

[0244] 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 sequences.

[0245] 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 2F9 or an antigen-binding fragment thereof having the same H-FR1-4 sequences.

[0246] 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 thereof having the same H-FR1-4.

[0247] 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 thereof having the same H-FR1-4.

[0248] 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 thereof having the same H-FR1-4.

[0249] 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 thereof having the same H-FR1-4.

[0250] 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 thereof having the same H-FR1-4.

[0251] 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 thereof having the same H-FR1-4.

[0252] 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 thereof having the same H-FR1-4.

[0253] 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 thereof having the same H-FR1-4 sequences.

[0254] 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 NO: 72 to SEQ ID NO: 84.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] In the present application, the isolated antigen binding protein can compete with the reference antibody for binding to human AFP 158-166 / HLA-A02*01* complex.

[0269] In the present application, the reference antibody may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.

[0270] In the present application, the HCDR3 of the reference antibody may comprise the amino acid sequence shown 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 HCDR3 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0271] In the present application, the HCDR3 of the reference antibody may comprise the amino acid sequence shown 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. For example, the HCDR3 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0272] In the present application, the HCDR2 of the reference antibody may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0273] In the present application, the HCDR2 of the reference antibody may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0274] In the present application, the HCDR1 of the reference antibody may comprise the amino acid sequence shown 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. For example, the HCDR1 sequence of the reference antibody may be defined according to the IMGT numbering system.

[0275] In the present application, the HCDR1 of the reference antibody may comprise the amino acid sequence shown 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 sequence of the reference antibody may be defined according to the Kabat numbering system.

[0276] For example, the HCDR1 of the reference antibody may comprise the amino acid sequence of 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 of 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 of 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.

[0277] In the present application, the reference antibody comprises a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR3 comprises the amino acid sequence of 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 of 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 of 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.

[0278] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0279] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0280] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0281] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0282] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0283] For example, the 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 include Antibody No. 3 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3).

[0284] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0285] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0286] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0287] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0288] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0289] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0290] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0291] Chimeric antigen receptors and modified cells

[0292] 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.

[0293] In the present application, the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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 HCDR3 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the IMGT numbering system.

[0294] In the present application, the HCDR3 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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. For example, the HCDR3 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the Kabat numbering system.

[0295] In the present application, the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the IMGT numbering system.

[0296] In the present application, the HCDR2 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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. For example, the HCDR2 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the Kabat numbering system.

[0297] In the present application, the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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. For example, the HCDR1 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the IMGT numbering system.

[0298] In the present application, the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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 sequence of the targeting portion of the chimeric antigen receptor may be defined according to the Kabat numbering system.

[0299] For example, the HCDR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown 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 shown 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 shown 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 can be defined according to the IMGT numbering system.

[0300] 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 of 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 of 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 of 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 targeting portion of the chimeric antigen receptor can be defined according to the Kabat numbering system.

[0301] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody 1B3 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0302] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody 1C4 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0303] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody 1C11 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0304] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody 1D12 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0305] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody 2F9 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0306] For example, the HCDR1 of the targeting moiety 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 moiety of the chimeric antigen receptor may comprise antibody No. 3 or an antigen-binding fragment thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0307] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0308] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0309] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0310] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0311] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0312] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0313] For example, the 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 thereof having the same HCDR3 (e.g., having the same HCDR1-3) therewith.

[0314] For example, the VH targeting portion of the chimeric antigen receptor may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.

[0315] In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 58 to SEQ ID NO: 65.

[0316] 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.

[0317] In the present application, the 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.

[0318] In the present application, the 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.

[0319] In the present application, the H-FR1 of the targeting portion of the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:65; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:66; the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:67 to SEQ ID NO:69; and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:71.

[0320] In the present application, the 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.

[0321] In the present application, the 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 thereof having the same H-FR1-4.

[0322] In the present application, the 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.

[0323] In the present application, the 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.

[0324] In the present application, the 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.

[0325] In the present application, the 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 thereof having the same H-FR1-4.

[0326] In the present application, the 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 thereof having the same H-FR1-4.

[0327] In the present application, the 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 thereof having the same H-FR1-4.

[0328] In the present application, the 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 thereof having the same H-FR1-4.

[0329] In the present application, the 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 thereof having the same H-FR1-4.

[0330] In the present application, the 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 thereof having the same H-FR1-4.

[0331] In the present application, the 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 thereof having the same H-FR1-4.

[0332] In the present application, the 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 thereof having the same H-FR1-4.

[0333] In the present application, the heavy chain variable region of the targeting portion of the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 72 to SEQ ID NO: 84.

[0334] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:72. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody 1B3 or a chimeric antigen receptor having the same heavy chain variable region.

[0335] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:73. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody 1C4 or a chimeric antigen receptor having the same heavy chain variable region.

[0336] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:74. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody 1C11 or a chimeric antigen receptor having the same heavy chain variable region.

[0337] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:75. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody 1D12 or a chimeric antigen receptor having the same heavy chain variable region.

[0338] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:76. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody 2F9 or a chimeric antigen receptor having the same heavy chain variable region.

[0339] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:77. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 3 or a chimeric antigen receptor having the same heavy chain variable region.

[0340] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:78. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 4 or a chimeric antigen receptor having the same heavy chain variable region.

[0341] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:79. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 7 or a chimeric antigen receptor having the same heavy chain variable region.

[0342] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:80. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 8 or a chimeric antigen receptor having the same heavy chain variable region.

[0343] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:81. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 15 or a chimeric antigen receptor having the same heavy chain variable region.

[0344] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:82. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 17 or a chimeric antigen receptor having the same heavy chain variable region.

[0345] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:83. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 24 or a chimeric antigen receptor having the same heavy chain variable region.

[0346] In the present application, the targeting portion of the chimeric antigen receptor 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 targeting portion of the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO:84. For example, the targeting portion of the chimeric antigen receptor may comprise the targeting portion of antibody No. 29 or a chimeric antigen receptor having the same heavy chain variable region.

[0347] 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% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology 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.

[0348] In the present application, the chimeric antigen receptor may include a transmembrane domain. For example, the transmembrane domain may include but is not limited to a transmembrane domain or a combination thereof derived from the following proteins: 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. For example, the transmembrane domain may include 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.

[0349] For example, the hinge region and transmembrane region may comprise the amino acid sequence set forth in SEQ ID NO: 128. For example, the hinge region and transmembrane region 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 region and transmembrane region may comprise the nucleotide sequence set forth in SEQ ID NO: 127.

[0350] In the present application, the chimeric antigen receptor may include a costimulatory signaling domain. For example, the costimulatory signaling domain may include, but is not limited to, a costimulatory signaling domain or a combination thereof 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 of CD83, CD40, and MyD88. For example, the costimulatory signaling domain may include a costimulatory signaling domain derived from 4-1BB. For example, the costimulatory signaling domain may include an amino acid sequence as shown 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.

[0351] For example, the costimulatory signaling domain may include a costimulatory signaling domain derived from CD28. For example, the costimulatory signaling domain may include an amino acid sequence as shown in SEQ ID NO: 130. For example, the costimulatory signaling domain may include 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 homology to the amino acid sequence shown in SEQ ID NO: 130. For example, the encoding costimulatory signaling domain may include a nucleotide sequence of SEQ ID NO: 129.

[0352] 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 PBj14Nef, Kaposi's sarcoma 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 shown in SEQ ID NO: 134. For example, the intracellular 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: 134. For example, the nucleotide sequence encoding the intracellular signaling domain may comprise the nucleotide sequence of SEQ ID NO: 133.

[0353] For example, the N-terminus of the transmembrane domain can be linked to the C-terminus of the targeting moiety. For example, the C-terminus of the transmembrane domain can be linked to the N-terminus of the costimulatory signaling domain. For example, the C-terminus of the costimulatory signaling domain can be linked to the N-terminus of the intracellular signaling domain.

[0354] 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 co-stimulatory signaling domain, and an intracellular signaling domain.

[0355] For example, the chimeric antigen receptor may comprise the following domains from N-terminus to C-terminus: an antigen binding protein of the present application (e.g., VHH), a transmembrane domain derived from CD8, a costimulatory signaling domain derived from CD137, and an intracellular signaling domain derived from CD3zeta.

[0356] In the present application, the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NO: 112 to SEQ ID NO: 124. For example, the chimeric antigen receptor 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 homology to the amino acid sequence shown in SEQ ID NO: 112 to SEQ ID NO: 124.

[0357] In another aspect, the present application provides a modified cell, wherein the modified cell may comprise the aforementioned chimeric antigen receptor. In the present application, the modification may comprise upregulating the expression of low-density lipoprotein receptor-related protein or a fragment thereof in the cell.

[0358] Low-density lipoprotein receptor-related protein or its fragments

[0359] In the present application, the low-density lipoprotein receptor-related protein may include one or more selected from the following group: low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0360] In the present application, the low-density lipoprotein receptor-related protein or its fragment can be derived from mammals. For example, it can be derived from humans, macaques, rats and mice. For example, the low-density lipoprotein receptor-related protein or its fragment can be derived from humans.

[0361] In the present application, the functional fragment may include a fragment or truncation of the low-density lipoprotein receptor-related protein that has 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 its truncation, and / or low-density lipoprotein receptor-related protein 5 and its truncation.

[0362] 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 the domain of a protein located within the cell membrane. In the present application, the intracellular domain may refer to the domain of the low-density lipoprotein receptor-related protein within the cell membrane. In the present application, the intracellular region may include the sequence from positions 24 to 243 of SEQ ID NO: 140 or an amino acid sequence having at least 80% homology thereto, or, include the sequence from positions 24 to 231 of SEQ ID NO: 144 or an amino acid sequence having at least 80% homology thereto. For another example, the truncated form of low-density lipoprotein receptor-related protein 6 may include 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 include the transmembrane region of low-density lipoprotein receptor-related protein 5 and the LDLR region of low-density lipoprotein receptor-related protein 5. In this application, the term "LDLR region" generally refers to the domain of the low-density lipoprotein receptor-related protein near the N segment outside the transmembrane region. This domain may have the function of enhancing Wnt signaling. In this application, the LDLR region may include amino acid sequences from positions 5 to 119 of SEQ ID NO: 138 or an amino acid sequence having at least 80% homology thereto, or sequences from positions 4 to 119 of SEQ ID NO: 142 or an amino acid sequence having at least 80% homology thereto.

[0363] In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof comprises an amino acid sequence shown in any one of the following or an amino acid sequence having at least 80% homology thereto: SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142 and SEQ ID NO: 144.

[0364] In the present application, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or its fragment comprises the nucleic acid sequence shown in any one of the following or a nucleic acid sequence having at least 80% homology thereto: SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141 and SEQ ID NO: 143.

[0365] In the present application, the modified cells may comprise immune cells (e.g., lymphocytes). In the present application, the modified cells may comprise modified T cells. In the present application, the modified cells may comprise modified memory stem cell-like T cells (TSCM) and / or genetically modified central memory T cells (TCM). In the present application, the TSCM may comprise CCR7+ and / or CD62L+. In the present application, the TSCM further comprises one or more properties selected from the group consisting of CD45RA+ or CD45RA-, CD45RO+ or CD45RO-, CD27+, CD28+, CD127+, CD122+, CD3+, CD4+, and CD8+.

[0366] In the present application, the modified cells may express the chimeric antigen receptor (CAR). For example, the modified cells may comprise a vector capable of expressing the chimeric antigen receptor (CAR). The vector may comprise a nucleotide molecule encoding the chimeric antigen receptor (CAR). For another example, the vector may be selected from the group consisting of a retroviral vector, a lentiviral vector, and / or a transposon plasmid.

[0367] In the present application, the vector capable of expressing the low-density lipoprotein receptor-related protein or its fragment and the vector capable of expressing the chimeric antigen receptor (CAR) can be the same vector or different vectors, as long as the one or more vectors can express the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR), so that the genetically modified immune cells simultaneously possess the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR).

[0368] For example, the vector capable of expressing the low-density lipoprotein receptor-related protein or its fragment and the vector capable of expressing the chimeric antigen receptor (CAR) can be the same vector. In this vector, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment and the nucleotide molecule encoding the chimeric antigen receptor (CAR) can be located in the same expression frame. For example, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment can be located at the 3' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).

[0369] In the present application, the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment can be directly or indirectly connected to the nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the indirect connection can be connected by a connecting sequence. The 5' end of the connecting sequence can be connected to the 3' end of the nucleotide molecule encoding the low-density lipoprotein receptor-related protein or its fragment, and the 3' end of the connecting sequence can be connected to the 5' end of the nucleotide molecule encoding the chimeric antigen receptor (CAR).

[0370] In the present application, the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR) expressed by the modified immune cells can be two independent proteins. That is, the two do not have any interconnected relationship to form any form of di (multi) polymer or protein complex. However, the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor (CAR) expressed by the genetically modified immune cells can also be interconnected. For example, in some cases, the two proteins formed by translation are not completely cut, and the low-density lipoprotein receptor-related protein or its fragment and the chimeric antigen receptor form a complex.

[0371] The chimeric antigen receptor may comprise a 2A sequence. The term "2A sequence" generally refers to a protease-independent self-cleavage amino acid sequence. The 2A sequence may facilitate transcription to produce two proteins. In the present application, the 2A sequence may include the sequence from positions 1 to 54 of SEQ ID NO: 136. For example, the nucleotide sequence encoding the 2A sequence may include the nucleotide sequence of SEQ ID NO: 135.

[0372] 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.

[0373] Peptides and immunoconjugates

[0374] On the other hand, the application provides one or more polypeptides, which may comprise the isolated antigen-binding proteins of the application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide may comprise a multispecific antibody (e.g., a bispecific antibody).

[0375] On the other hand, the present application provides one or more immunoconjugates, which may comprise the isolated antigen-binding protein of the present application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, a marker and / or a detection agent.

[0376] Nucleic acids, vectors, and cells

[0377] In another aspect, the present application further provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding protein or polypeptide described herein. For example, each of the one or more nucleic acid molecules can encode the entire antigen-binding protein or a portion thereof (e.g., one or more of HCDRs 1-3 or a heavy chain variable region).

[0378] For example, when the nucleic acid molecules respectively encode a portion of the antigen-binding protein or polypeptide, the products encoded by the nucleic acid molecules can be combined to form a functional (eg, capable of binding to AFP) isolated antigen-binding protein of the present application.

[0379] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology.

[0380] In the present application, nucleic acids encoding the isolated antigen-binding protein can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain nucleic acid molecules of the isolated antigen-binding protein or polypeptide described in the present application.

[0381] In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vectors may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Furthermore, the vectors may also contain expression control elements that allow for proper 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 certain embodiments, the expression control sequences are adjustable elements. The specific structure of the expression control sequences may vary depending on the function of the species or cell type, but typically include 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence functionally linked to a nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a portion of the promoter may be fused to a portion of a promoter of a gene of another cellular protein (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not include additional introns. One or more nucleic acid molecules described herein may be operably linked to the expression control element.

[0382] The vector may include, for example, plasmids, clays, viruses, phages or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. The viral vector may be directly administered to the patient (in vivo) or may be administered in an indirect manner, for example, using a virus-treated cell in vitro and then administering the treated cell to the patient (ex vivo). Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, slow virus vectors, adenovirus vectors, adeno-associated virus vectors and herpes simplex virus vectors for gene transfer. In some cases, gene transfer may be integrated into the host genome using a retrovirus, slow virus and adeno-associated virus method to express the inserted gene for a long time. Slow virus vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce higher viral titers. The lentiviral vector may comprise a long terminal repeat 5'LTR and a truncated 3'LTR, RRE, a rev response element (cPPT), a central termination sequence (CTS) and / or a post-translational regulatory element (WPRE).The vector described in the present application can be introduced into cells.

[0383] On the other hand, the application provides a kind of cell. The cell may comprise the separated antigen-binding protein described in the application, the polypeptide, the immunoconjugate, one or more nucleic acid molecules and / or one or more vectors described in the application. For example, each or each cell may comprise one or a nucleic acid molecule or vector described in the application. For example, each or each cell may comprise multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described in the application. For example, the vector described in the application may be introduced into the host cell, such as a prokaryotic cell (e.g., bacterial cell), a CHO cell, a NS / 0 cell, a HEK293T cell, a 293F cell or a HEK293A cell, or other eukaryotic cells, such as cells from plants, fungi or yeast cells, etc. The vector described in the application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the cell may comprise a yeast cell. For example, the cell may comprise an Escherichia coli cell. For example, the cell may comprise a mammalian cell. For example, the cell may comprise an immune cell.

[0384] The cell may include an immune cell. In some cases, the cell may include an immune cell. For example, the cell may include a T cell, a B cell, a natural killer (NK) cell, a macrophage, an NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and / or a peripheral blood mononuclear cell. For example, the cell may include a T cell.

[0385] Pharmaceutical composition

[0386] In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen-binding protein, polypeptide, immunoconjugate, isolated nucleic acid molecule, vector, cell, and / or pharmaceutically acceptable adjuvant and / or excipient described herein. In the present application, the pharmaceutically acceptable adjuvant may include a buffer, antioxidant, preservative, low molecular weight polypeptide, protein, hydrophilic polymer, amino acid, sugar, chelating agent, counterion, metal complex, and / or nonionic surfactant. Unless incompatible with the cells described herein, any conventional medium or reagent may be considered for use in the pharmaceutical composition of the present application. In the present application, the pharmaceutically acceptable excipient may include additives other than the main drug in a pharmaceutical formulation, also referred to as an excipient. For example, the excipient may include a binder, filler, disintegrant, or lubricant in a tablet. For example, the excipient may include wine, vinegar, or medicinal juice in a traditional Chinese medicine pill. For example, the excipient may include the matrix component of a semisolid ointment or cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0387] Kits, uses and methods

[0388] In another aspect, the present application provides a method for detecting or measuring AFP, which may include using the isolated antigen-binding protein or the polypeptide.

[0389] In the present application, the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.

[0390] For example, the method may include a method for detecting the presence and / or amount of AFP for non-diagnostic purposes, which may include the following steps:

[0391] 1) contacting the sample with the antigen-binding protein of the present application; and

[0392] 2) detecting the presence and / or amount of the antigen binding protein bound to the sample to determine the presence and / or expression level of AFP in the sample obtained from the subject.

[0393] On the other hand, the present application provides an AFP kit, which may include and use the isolated antigen-binding protein or the polypeptide.

[0394] In the present application, the kit may further comprise instructions for use, which describe methods for detecting the presence and / or content of AFP. For example, the methods may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0395] In another aspect, the present application provides a use of the isolated antigen-binding protein or polypeptide in preparing a kit, wherein the kit can be used for a method for detecting the presence and / or content of AFP. For example, the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.

[0396] On the other hand, the present application provides an isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and the pharmaceutical composition for preventing, alleviating and / or treating a disease or condition.

[0397] On the other hand, in the present application, the kit and / or the pharmaceutical combination is used to prevent, alleviate and / or treat a disease or condition.

[0398] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression of AFP in the tumor microenvironment or in tumor cells is upregulated compared to normal cells. The tumor associated with the protein expression of AFP may be an AFP-positive tumor. In an AFP-positive tumor, the protein expression of AFP in tumor cells or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that in normal cells. For example, the tumor may include a solid tumor. For example, the tumor may include liver cancer.

[0399] On the other hand, the present application provides a 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 preparation of a drug for preventing, alleviating and / or treating a disease or condition.

[0400] In another aspect, the present application provides a use of a drug combination in the preparation of a medicament for preventing, alleviating and / or treating a disease or condition.

[0401] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression of AFP in the tumor microenvironment or in tumor cells is upregulated compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that in normal cells.

[0402] For example, the tumor may include a solid tumor. For example, the tumor may include liver cancer.

[0403] On the other hand, the present application provides a method for preventing and / or treating a disease or condition, which comprises 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.

[0404] For example, the disease or condition may include a tumor. For example, the tumor may include a tumor associated with the expression of AFP. The term "tumor associated with the expression of AFP" generally refers to an altered expression of AFP in the tumor microenvironment or in tumor cells compared to normal cells. For example, the "tumor associated with the expression of AFP" may be a tumor in which the expression of AFP in the tumor microenvironment or in tumor cells is upregulated compared to normal cells. The tumor associated with AFP protein expression may be an AFP-positive tumor. In AFP-positive tumors, the protein expression of AFP in tumor cells or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that in normal cells.

[0405] For example, the tumor may include a solid tumor. For example, the tumor may include liver cancer.

[0406] The pharmaceutical compositions and methods described herein can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, etc. The pharmaceutical compositions and methods described herein can be used for other disease conditions that rely on immune responses, such as inflammation, immune diseases, and infectious diseases.

[0407] 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. Furthermore, the non-human animal may include any animal species other than humans, such as livestock, rodents, primates, domestic animals, or poultry. The human may be Caucasian, African, Asian, Semitic, or of other races, or a hybrid of various races. For another example, the human may be elderly, an adult, a teenager, a child, or an infant.

[0408] The effective dose in humans can be extrapolated from the effective dose in experimental animals. For example, Freireich et al. described the relationship between animal and human doses (based on milligrams per square meter of body surface) (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).

[0409] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen binding protein, chimeric antigen receptor, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0410] Example

[0411] Example 1 Construction of a synthetic nanobody (VHH antibody) library and screening of anti-AFP / HLA02 TCR-like antibodies

[0412] 1.1 Construction of artificially synthesized nanoantibody (VHH antibody) library

[0413] By comparing the nucleotide sequences and protein structures of 296 non-repetitive nanobodies (VHH antibodies) from the PDB (Protein Data Bank) database, we determined the mutation strategy for the CDRs of the synthetic nanobody library. Three CDR3 lengths were selected: 14, 17, and 21 amino acids. The commercially available nanobody, Caplacizumab (publication number: NL300966I2), was selected as the backbone to synthesize the full nucleotide sequence of the nanobody Caplacizumab. The synthetic full nucleotide sequence of Caplacizumab was cloned into the HP153 phage vector. Single-stranded DNA from the HP153 phage vector was then extracted and used as a template to mutate the nucleotides in the CDRs of the nanobody Caplacizumab using the Kunkel mutagenesis method. A double-stranded DNA library of the mutated nanobodies was generated. The double-stranded DNA was electroporated into competent Escherichia coli SS320 cells pre-infected with M13KO7. After overnight incubation, the phage supernatant was collected, ultimately constructing a library with a diversity of 1.44×10 10 The artificial synthetic nanoantibody library NanoOri_1.0 (Yuanqi Biotechnology (Shanghai) Co., Ltd.) was used as a seed library for antibody sequence screening.

[0414] 1.2 Liquid-phase panning of synthetic nanoantibody libraries

[0415] Spread TG1 Escherichia coli competent cells on a bacterial culture plate without antibiotics. The next day, take 15 ml of antibiotic-free 2×YT medium and inoculate a single TG1 colony. Culture at 37°C overnight. Take new antibiotic-free 2×YT medium and inoculate 200 μl of the overnight cultured TG1 bacterial solution. Culture the TG1 bacterial solution at 37°C and 250 rpm until OD600 = 0.8. Take 900 μl of magnetic beads with streptavidin and add them to three 1.5 ml EP tubes, 300 μl per tube, place them on a magnetic stand, and wash them three times with PBS. Add 30 μg of biotin-labeled human TERT540 peptide to two of the EP tubes, respectively, and record them as 540-1 and 540-2. Add 30 μg of biotin-labeled human AFP peptide to the third tube, record them as hAFP. The EP tube was incubated at room temperature for 1 hour, placed on a magnetic rack, and washed 3 times with PBS; 700 μl of phage library was added to the tube of 540-1, incubated at room temperature for 1 hour, placed on a magnetic rack, and the supernatant was taken; the supernatant was added to the tube of hAFP, 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 phage, and the tube was allowed to stand at room temperature for 15 minutes, placed on a magnetic rack, and the supernatant was taken. Then 250 μl of Tris-HCl (pH 8.0) was added to the supernatant for neutralization; the neutralized supernatant was added to the tube of 540-2, incubated at room temperature for 1 hour, placed on a magnetic rack, and the supernatant was taken; the secondary library was obtained; in the secondary 20 ml of the previously prepared TG1 bacterial solution (OD600 = 0.8) was added to the library and allowed to stand at 37°C for 30 minutes. 1 ml was sampled and counted. 40 μl of helper phage M13K07 was added and allowed to stand at 37°C for 30 minutes. The antibiotics ampicillin and kanamycin were added, along with 20 ml of 2×YT medium, and the cells were incubated overnight at 37°C and 220 rpm. The overnight culture was centrifuged at 8000 rpm, the supernatant removed, and precipitated with 1 / 5 the volume of PEG-NaCl. The cells were incubated on ice for 1 hour and centrifuged at 8000 rpm for 30 minutes. The supernatant was removed and the pellet resuspended in 1 ml of PBS. This constituted the first round of panning, and the phage was then used for the second round of panning. The selection pressure was adjusted in each subsequent round to achieve the desired target. Specific conditions are shown in Table 1. A total of five rounds of panning were completed. The panning results are shown in Table 1. Significant enrichment began to occur in the third round. The ELISA results from the five rounds of panning are shown in Figure 1. The secondary antibody used was M13 phage antibody (HRP) (manufacturer: Sino Biological).

[0416] Table 1 Nanobody panning conditions and results

[0417]

[0418] Monoclonal clones were selected for sequencing identification and analysis; sequence alignment analysis was performed using BioEdit software, and the statistical results are shown in Figure 2. Among the selected monoclonal clones, there were a total of 13 repeated VHH antibody 1B3 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 1), 11 repeated VHH antibody 1C4 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 2), 5 repeated VHH antibody 1C11 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 3), 4 repeated VHH antibody 1D12 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 4), and 4 repeated VHH antibody 2F9 nucleotide sequences (nucleotide sequence shown in SEQ ID NO: 5). There were 8 non-repeated VHH antibody single nucleotide sequences.

[0419] Example 2 Expression and Characterization of Candidate VHH-Fc Antibodies

[0420] 2.1 Construction of eukaryotic expression vectors for candidate VHH-Fc antibodies and antibody purification

[0421] Primers were designed to perform PCR amplification on the candidate VHH antibody nucleotide sequence obtained in Example 1 (nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13). The nucleotide sequences were then cloned by homologous recombination into the ampicillin-resistant pcDNA3.4 vector double-digested with the restriction endonucleases SfiI and NotI. The 3' end of the VHH antibody nucleotide sequence was ligated to the 5' end of the antibody Fc portion (SEQ ID NO: 125), thereby constructing a candidate VHH-Fc antibody eukaryotic expression vector. A map of the constructed vector is shown in FIG5 . The correctly sequenced recombinant plasmid was 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 shows, from left to right, a 100 bp marker, 1B3 (VHH antibody 1B3 nucleotide), 1C4 (VHH antibody 1C4 nucleotide), 1C11 (VHH antibody 1C11 nucleotide), 1D12 (VHH antibody 1D12 nucleotide), and 2F9 (VHH antibody 2F9 nucleotide) (approximately 400 bp). The electrophoresis diagram of the purified protein is shown in Figure 4 , which shows, from left to right, a marker, reduced 1B3 (VHH-Fc antibody containing the VHH antibody 1B3 nucleotide sequence), and non-reduced 1B3. The molecular weight of the purified 1B3 is correct (approximately 41 kDa after reduction).

[0422] 2.2 Identification of affinity of candidate VHH-Fc antibodies

[0423] Using the ForteBio AHC sensor, 100 nM candidate VHH-Fc antibodies were first bound, and then bound to the human AFP158-166 / HLA-A02*01* or human TERT540 / HLA-A02*01* complex, respectively. The starting concentration was 150 nM, 2-fold dilution, 7 concentration gradients, binding for 5 minutes, and dissociation for 10 minutes. The affinity data were analyzed using Data Analysis 9.0. The data results are shown in Table 2. The affinity results are shown in Figure 6. Among them, No. 17 (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 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 24, the antibody nucleotide sequence is shown in SEQ ID NO: 12) did not dissociate substantially. No. 3 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 3, the antibody nucleotide sequence is shown in SEQ ID NO: NO: 6) has an affinity that is about 9 times higher than that of the Eureka positive control antibody Ab61 (amino acid sequence is SEQ ID NO: 86, nucleotide sequence is SEQ ID NO: 85, Fc amino acid sequence is shown in SEQ ID NO: 88, and Fc nucleotide sequence is shown in SEQ ID NO: 87). No. 4 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 4, the antibody nucleotide sequence is shown in SEQ ID NO: 7), No. 29 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody No. 29, the antibody nucleotide sequence is shown in SEQ ID NO: 13), 1B3 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1B3, the antibody nucleotide sequence is shown in SEQ ID NO: 1) and 1C4 (VHH-Fc antibody containing the nucleotide sequence of VHH antibody 1C4, the antibody nucleotide sequence is shown in SEQ ID NO: 2) also have affinities that are better than those of the Eureka positive control antibody Ab61.

[0424] Table 2 Nanobody affinity test results

[0425]

[0426] 2.3 Identification of non-specific binding activity of candidate VHH-Fc antibodies to different endogenous human peptides

[0427] The binding activity of the antibodies was detected using an iQue Screener flow cytometer. Specifically, T2 cells were HLA-A02*01* positive cells and loaded with human AFP. 158polypeptide (amino acid sequence as shown in SEQ ID NO: 90, nucleic acid sequence as shown in SEQ ID NO: 89), human NY-Eso-1 157 polypeptide (amino acid sequence as shown in SEQ ID NO: 91), and IFI30 (amino acid sequence as shown in any one of SEQ ID NOs: 92-95), BTG2 (amino acid sequence as shown in SEQ ID NO: 96), BCR (amino acid sequence as shown in SEQ ID NO: 97), SSR1 (amino acid sequence as shown in any one of SEQ ID NOs: 98-99), PPP2R1B (amino acid sequence as shown in SEQ ID NO: 100), DDX5 (amino acid sequence as shown in SEQ ID NO: 101), CTSG (amino acid sequence as shown in SEQ ID NO: 102), CD247 (amino acid sequence as shown in SEQ ID NO: 103), DMTN (amino acid sequence as shown in SEQ ID NO: 104), CALR (amino acid sequence as shown in SEQ ID NO: 105), PIM1 (amino acid sequence as shown in SEQ ID NO: 106), HLA-E (amino acid sequence as shown in SEQ ID NO: 107). 20 human endogenous polypeptides were tested, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 90, 81, 82, 83, 84, 57, 58, 69, 71, 72, 85, 73, 75, 86, 77, 87, 90, 81, 83, 85, 77, 88, 89, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 100, 100, 100, 100, 100, 100 6 Add 30 μl of cells to each well of a 96-well pointed bottom plate to a volume of 3 × 10 cells / ml. 4The detection antibody was prepared using a buffer solution, starting at 10 μg / ml, and the antibody was diluted 3-fold. Seven concentration gradients were set up and detected using a flow cytometer to further identify the specific binding activity of the candidate VHH antibodies. As 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) had 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 Eureka's positive control antibody Ab61 all had 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 peptide and human endogenous mixed peptide / T2.

[0428] 2.4 Identification of cross-reactivity of candidate VHH-Fc antibodies with mouse AFP158 peptide (amino acid sequence shown in SEQ ID NO: 126)

[0429] Further characterization of cross-reactivity with the mouse AFP158 peptide / HLA-A*02:01 complex on the surface of living cells. The mouse AFP158 peptide differs from the human AFP158 peptide at two amino acid positions, position 4 and position 9. Antibodies that cross-react with both human and mouse AFP158 peptide / MHC complexes are suitable for evaluating antibody drug toxicity in HLA-A02*01* transgenic mice. As shown in Figure 7C, all five candidate VHH-Fc antibodies (containing the nucleotide sequences of VHH antibodies 1C11, 1C4, 2F9, 1B3, and 1D12) bound to mouse AFP158 / T2 cells, while Eureka's positive control antibody, Ab61, showed weaker binding activity.

[0430] Example 3 Construction of CAR core plasmid containing candidate VHH antibody nucleotide sequence, lentiviral packaging and CAR-T cell preparation

[0431] 3.1 Construction of CAR core plasmid containing VHH antibody nucleotide sequence

[0432] The core plasmid was double-digested with restriction endonucleases SphI and NotI and mixed with candidate VHH antibody nucleotide sequences at a ratio of 1:3 (molar ratio) for homologous recombination. The nucleotide sequence of the homologously recombined core plasmid was verified by sequencing, and the amplified plasmid was stored for future 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.

[0433] 3.2 Lentiviral packaging and titer determination

[0434] The vector system used to construct the lentiviral plasmid of the present invention belongs to the third generation lentiviral vector system. The system has three plasmids, namely: 1: packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein; 2: PMD2.G plasmid encoding envelope protein VSV-G; 3: CAR core plasmid constructed by 3.1 containing nucleotide sequence encoding VHH antibody. In the core plasmid based on the BBz platform plasmid, the gene encoding CAR is expressed by the elongation factor-1α (EF-1α) promoter. The packaging process of the lentivirus is as follows: 1×10 6 293T cells were suspended in 2 ml of 10% FBS DMEM medium, plated in a single well of a 6-well plate, and cultured overnight; 144 ul of medium (50 times the mass of the plasmid) was aspirated and mixed with 144 ul 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, and the cells were gently mixed and cultured at 37°C in a CO2 incubator for 12 hours; the medium containing the plasmid was removed, the cells were washed once with PBS, and then replaced with 2 ml of DMEM medium containing 5% FBS and cultured for 48 hours; 2.5 ml of viral supernatant was collected, centrifuged at 3000 rpm for 5 minutes, aliquoted and frozen at -80°C until use, and the titer was detected.

[0435] 3.3 Preparation of CAR-T cells containing VHH antibodies

[0436] The method for producing CAR-T cells containing VHH antibodies is as follows: human peripheral blood mononuclear cells are obtained by density gradient centrifugation; the peripheral blood mononuclear cells are resuspended in medium containing 200 U / ml interleukin-2 to a cell density of 2×10 6 / ml, CD3 / CD28 magnetic beads were added at a ratio of 1:3 (cells: magnetic beads) to activate T cells; the activated peripheral blood mononuclear cells were cultured in a 37°C CO2 incubator for 24 hours; the above-obtained lentiviral supernatant was added at a virus infection multiplicity (MOI) of 3, 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; the well plate was returned to a 37°C CO2 incubator and cultured for 24 hours; centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells were resuspended in fresh X-VIVO medium (manufacturer: LONZA) containing 500 U / ml interleukin 2 to a cell density of 0.6×10 6 / ml, and cultured in a 37°C CO2 incubator; cells were counted every 2 days, and fresh X-VIVO medium (manufacturer: LONZA) containing 500U / ml interleukin-2 was added to adjust the cell density back to 0.6×10 6 / ml; CAR-T cells cultured for 9-14 days detect cell positivity: The lentivirus used to infect cells carries GFP. After the lentivirus infects the cells, the GFP positivity rate is detected by flow cytometry to obtain the CAR expression positivity rate. Cells with a positivity rate >20% can be used for tumor killing experiments.

[0437] Example 4 Construction and detection of overexpression cell lines HepG2-MiniG cells and SK-HEP-1-MiniG cells

[0438] Human AFP was injected into the cell membrane by lentiviral packaging and lentiviral infection. 158-166The gene (SEQ ID NO: 89) was introduced into HepG2-MiniG and SK-HEP-1 cells, respectively, to prepare HepG2-MiniG and SK-HEP-1-MiniG cells. Taking the construction of SK-HEP-1-MiniG cells as an example, 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:). The PCR and enzyme digestion results are shown in Figure 10. The PCR results of the human AFP158-166 nucleotide sequence amplification are as follows (A): from left to right, 100bp Marker, human AFP158-166 band (about 150bp); the vector enzyme digestion results are as follows (B): Marker IV, band after vector digestion (approximately 7500 bp); After lentiviral infection, the recombinant plasmid pLV-C-GFPS-AFP-short was transfected into SK-HEP-1 cells. Monoclonal SK-HEP-1-MiniG cells were selected for flow cytometry binding activity assays, as shown in Figure 11. Monoclonal SK-HEP-1-MiniG-7 (SK-HEP-1-MiniG cell monoclonal clone 7) had the highest mean fluorescence intensity and was expanded and used as target cells. HepG2-MiniG cells were constructed using the same method as SK-HEP-1-MiniG cells. Flow cytometry analysis of the positive rates of HepG2-MiniG and SK-HEP-1-MiniG cell monoclonal clones after expansion was performed. The results are shown in Figure 12. The positive rates of HepG2-MiniG cells reached 87.5%, and those of SK-HEP-1-MiniG cells reached 93.9%.

[0439] Example 5 In vitro killing and factor detection of CAR-T cells containing candidate VHH antibodies

[0440] 5.1 Repeated in vitro stimulation experiment of CAR-T cells containing candidate VHH antibodies

[0441] CAR-T cells were prepared according to Example 3. On the 9th to 12th day of amplification, the positive rate was detected and the CAR-T cells were resuspended in serum-free X-VIVO medium (manufacturer: LONZA) to 4×10 5 / ml density as effector cells; HepG2-MiniG cells prepared in Example 4 were resuspended in serum-free X-VIVO medium (manufacturer: LONZA) to a density of 4×10 5 The target cells were mixed at a density of 1000 / ml at a ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator. The culture medium was observed every 2 days and 1 volume of culture medium was added when the culture medium turned from orange to yellow. The cells were counted on the 4th to 5th day to calculate the expansion times. After counting, 5×105 CAR-T cells were expanded again for the second, third, and fourth rounds using the same method as described above. The total cumulative expansion fold was calculated by multiplying the first round expansion fold by the second, third, and fourth round expansion folds.

[0442] The results of repeated stimulation are shown in Figure 13A. After three rounds of repeated stimulation, the cumulative expansion multiple of CAR-T cell 1B3 was comparable to that of CAR-T cell Ab61. As shown in Figure 13B, after four rounds of repeated stimulation, the cumulative expansion multiples of CAR-T cells No. 3, No. 4, No. 7, No. 8, No. 17, and No. 29 containing the candidate VHH antibody all exceeded that of CAR-T cell Ab61.

[0443] 5.2 In vitro killing assay of CAR-T cells containing candidate VHH antibodies

[0444] The SK-HEP-1-MiniG cells prepared in Example 4 were used as target cells, the CAR-T cells containing the candidate VHH antibody 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 process was as follows: the infection efficiency of the packaged CAR core plasmid lentivirus prepared in Example 3 was detected, and the infection ratio was adjusted to the same level in each group using T cells not infected with lentivirus; 200 μl of X-VIVO medium was added according to the effector cell: target cell (effect-target ratio) = 1:1, and the number of target cells was 1×10 4 / well, as the experimental group; wells containing only effector cells equal to the experimental group were used as the effector cell self-release background group; wells containing only target cells equal to the experimental group were used as the target cell self-release background group; the obtained cells were cultured in a 37°C CO2 incubator for 18 hours; 20μl of 10× lysis buffer was added to some wells containing only target cells and reacted for 45 minutes as the maximum release of target cells. The obtained cell culture well plates were centrifuged at 300g for 5 minutes, and 50μl of supernatant was collected for detection of the release of lactate dehydrogenase LDH. The detection method was referred to the instructions of the CytoTox96 non-radioactive cytotoxicity kit (manufacturer: Promega). The released LDH in the culture medium supernatant can be detected by a coupled enzyme reaction. The cell killing activity calculation formula is:

[0445] Toxicity % = 100 × (experimental group - effector cell self-release - target cell self-release + culture base value) / (maximum target cell release - target cell self-release).

[0446] According to the cell killing formula, the killing effect of CAR-T cells containing candidate VHH antibodies on target cells was analyzed, and candidate VHH antibodies with obvious killing effects were selected for in vivo functional evaluation.

[0447] The killing results are shown in Figure 13C, which show that the five candidate VHH antibodies have similar killing effects on HLA-A02*01*+ / AFP+ positive cells SK-HEP-1-MiniG. In the cell killing results with HepG2-MiniG as target cells, CAR-T cells containing the candidate VHH antibody 1C11 have better killing effects on target cells than CAR-T cells containing the positive control antibody Ab61, while CAR-T cells containing the candidate VHH antibody 2F9 have better killing effects on HepG2 cells with low expression levels of HLA-A02*01*+ / AFP+ than CAR-T cells containing the positive control antibody Ab61. As shown in Figure 13F, the results showed that the CAR-T cells containing candidate VHH antibodies 3, 4, 7, 8, 15, 17, 24, and 29 had comparable cell-killing effects on HLA-A02*01*+ / AFP+-positive SK-HEP-1-MiniG cells as those containing the positive control antibody Ab61. In the cell-killing results against HepG2-MiniG target cells, the CAR-T cells containing candidate VHH antibodies 3 and 4 had significantly better cell-killing effects than the CAR-T cells containing the positive control antibody Ab61.

[0448] 5.3 In vitro cytokine secretion assay of CAR-T cells containing candidate VHH antibodies

[0449] 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 antibody prepared in Example 3 were used as effector cells, and T cells not infected with the lentivirus were used as control effector cells. The specific experimental process is as follows: the infection efficiency of the packaged CAR core plasmid lentivirus prepared in Example 3 was detected, and the ratio of T cells not infected with the virus was adjusted to be consistent in each group; the effector cells: target cells (effect-target ratio) were added to 200 μl X-VIVO medium at a ratio of 1:1, and the number of target cells was 2×10 4 / well, as the experimental group; wells containing only effector cells equal to those in the experimental group served as the background group; the obtained cells were cultured in a 37°C CO2 incubator for 18 hours; the obtained cell culture well plates were centrifuged at 300g for 5 minutes, and 50μl of supernatant was collected for detection of IL2 and IFN-γ expression levels. The detection method was referred to the instructions of the R&D DuoSet ELISA kit (manufacturer: R&D SYSTEM).

[0450] 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 more IFN-γ. As shown in Figure 13E, all CAR-T cells containing candidate VHH antibodies had a low background level of IL-2 cytokines against HepG2-MiniG cells and virus-uninfected 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 released more IFN-γ. 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 had better IL2 cytokine secretion, and CAR-T cells with all candidate VHH antibodies had a low IL2 background against HepG2-MiniG cells and virus-uninfected T cells.

[0451] Example 6: Validation of VHH Antibody CAR-T Cell Efficacy in Vivo

[0452] CAR-T cells containing candidate VHH antibody 17 and CAR-T cells containing candidate VHH antibody 1B3 were randomly selected for in vivo efficacy evaluation. 6 mice were injected with 6×10 6 K-HEP-1-MiniG cells were used to implant the tumor subcutaneously in the middle back of the right upper limb. 3 At the same time, NDG mice were divided into 7 groups, and each group was injected with drugs via intratumoral or tail vein injection: 1 group of T cells not infected with the virus (control group IV), 2 groups injected with CAR-T cells containing positive control antibody Ab61 (Ab61-IT for intratumoral injection, Ab61-IV for tail vein injection), 2 groups injected with CAR-T cells containing candidate VHH antibody 1B3 (1B3-IT for intratumoral injection, 1B3-IV for tail vein injection), and 2 groups injected with CAR-T cells containing candidate VHH antibody 17 (17-IT for intratumoral injection, 17-IV for tail vein injection). Each group had 5 mice, and 1×10 7 The CAR-T cells were then infused back into the mice every two weeks. The body weight and tumor size of each group of mice were recorded after administration, and their biological responses were observed.

[0453] At PG (D52), the tumor volume and tumor inhibition rate of each group are shown in Table 3. The average tumor volume of the T cell group not infected with the virus was 973.98 ± 136.05 mm 3Compared with the virus-uninfected T cell group (control group IV), the average tumor volumes of mice in the CAR-T cell intratumoral injection group containing the positive control antibody Ab61 (Ab61-IT), the CAR-T cell intratumoral injection group containing the candidate VHH antibody 1B3 (1B3-IT), the CAR-T cell intratumoral injection group containing the candidate VHH antibody 17 (17-IT), the CAR-T cell tail vein injection group containing the positive control antibody Ab61 (Ab61-IV), the CAR-T cell tail vein injection group containing the candidate VHH antibody 1B3 (1B3-IV), and the CAR-T cell tail vein injection group containing the candidate VHH antibody 17 (17-IV) were 2.73±1.71mm, respectively. 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 3 ; Compared with the T cell group not infected with the virus (control group IV), there were significant differences (P<0.0001); the tumor volume inhibition rates were 109.68%, 109.57%, 108.88%, 107.75%, 107.19% and 105.67% respectively.

[0454] Table 3 Statistics of tumor volume and inhibition rate in PG (D52) mouse animal experiments

[0455]

[0456] The animal experiment process is shown in Figure 14. The results of the tumor growth curve in Figure 15A show that the tumors of each group of mice began to regress continuously on the 14th day after the first dose of CAR-T cells were infused. The average tumor volume of mice in each experimental group was significantly different from that in the control group (0.0001

Claims

1. An isolated antigen-binding protein having one or more of the following properties: 1) specifically binds to the human AFP158-166 / HLA-A02*01* complex with a KD value of approximately 3.1E-09 M or less; 2) Can bind to the mouse AFP158 / / HLA-A02*01* complex.

2. The isolated antigen-binding protein according to claim 1, comprising a HCDR3 comprising the amino acid sequence shown 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. The isolated antigen-binding protein according to any one of claims 1-2, comprising a HCDR2 comprising the amino acid sequence shown 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. The isolated antigen-binding protein according to any one of claims 1 to 3, comprising a HCDR1 comprising the amino acid sequence shown 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. The isolated antigen-binding protein according to any one of claims 1 to 4, comprising HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in any one of SEQ ID NO: 72 to SEQ ID NO:

84.

6. The isolated antigen-binding protein of any one of claims 1 to 5, comprising a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR3 comprises the amino acid sequence of 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 of 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 of 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 isolated antigen-binding protein of claim 6, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups: 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.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, comprising H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 58 to SEQ ID NO:

65.

9. The isolated antigen-binding protein according to any one of claims 1 to 8, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:

66.

10. The isolated antigen-binding protein according to 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 shown in any one of SEQ ID NO: 67 to SEQ ID NO:

69.

11. The isolated antigen-binding protein according to any one of claims 1 to 10, comprising H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 70 to SEQ ID NO:

71.

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

71.

13. The isolated antigen-binding protein of claim 12, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 comprise any one of the following amino acid sequences: 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.

14. The isolated antigen-binding protein according to any one of claims 1 to 13, comprising a heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO: 72 to SEQ ID NO:

84.

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

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

18. The isolated antigen binding protein of any one of claims 15-17, 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. The isolated antigen-binding protein according to any one of claims 1 to 18, comprising the amino acid sequence shown in any one of SEQ ID NO: 72 to SEQ ID NO:

84.

20. A chimeric antigen receptor comprising a targeting moiety comprising a HCDR3 comprising the amino acid sequence of 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. The chimeric antigen receptor of claim 20, wherein the targeting moiety comprises 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.

22. The chimeric antigen receptor of any one of claims 20-21, wherein the targeting moiety comprises HCDR1 comprising the amino acid sequence of 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 20-22, wherein the targeting moiety comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in any one of SEQ ID NO: 72 to SEQ ID NO:

84.

24. The chimeric antigen receptor of any one of claims 20-23, wherein the targeting moiety comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence of 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 of 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 of 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 chimeric antigen receptor of claim 24, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one of the following groups: 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.

26. The chimeric antigen receptor of any one of claims 20-25, wherein the targeting moiety comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:58 to SEQ ID NO:

65.

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

66.

28. The chimeric antigen receptor of any one of claims 20-27, wherein the targeting moiety comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and wherein the H-FR3 comprises the amino acid sequence of any one of SEQ ID NO: 67 to SEQ ID NO:

69.

29. The chimeric antigen receptor of any one of claims 20-28, wherein the targeting moiety comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:70 to SEQ ID NO:

71.

30. The chimeric antigen receptor of any one of claims 20-29, wherein the targeting moiety 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 NO:58 to SEQ ID NO: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 NO:67 to SEQ ID NO:69; and H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:70 to SEQ ID NO:

71.

31. The chimeric antigen receptor of claim 30, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 comprise any one of the following amino acid sequences: 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.

32. The chimeric antigen receptor of any one of claims 20-31, 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 20-33, 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 20-35, wherein the targeting moiety comprises the amino acid sequence shown in any one of SEQ ID NO: 72 to SEQ ID NO:

84.

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

38. The chimeric antigen receptor of claim 37, wherein the hinge region comprises a hinge region derived from the following proteins: IgG4, IgG1, and CD8.

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

147.

40. The chimeric antigen receptor of any one of claims 20-39, 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 of any one of claims 40-41, wherein the transmembrane domain comprises a transmembrane domain derived from CD8.

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

149.

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

45. The chimeric antigen receptor of any one of claims 20-44, comprising a co-stimulatory 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 of CD83, CD40, and MyD88, or a combination thereof.

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

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

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

130.

50. The chimeric antigen receptor of any one of claims 45-49, 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 20-50, 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 herpesvirus (HSKV), DAP10, and DAP-12, or a combination thereof.

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

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

134.

55. The chimeric antigen receptor of any one of claims 51-54, 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 according to any one of claims 20 to 55, comprising the amino acid sequence shown in any one of SEQ ID NO: 112 to SEQ ID NO:

124.

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

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

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

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

61. A modified cell comprising the isolated antigen binding protein of any one of claims 1-19, the chimeric antigen receptor of any one of claims 20-56, 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. The modified cell according to claim 61, wherein the modification comprises upregulating the expression of 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 fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

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

65. The modified cell of any one of claims 63-64, wherein the functional fragment comprises a fragment or truncation of the low-density lipoprotein receptor-related protein that has the activity of the low-density lipoprotein receptor-related protein.

66. The modified cell of any one of claims 62-65, 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. The modified cell of any one of claims 62-66, wherein the truncation of low-density lipoprotein receptor-related protein 6 comprises the intracellular region of the low-density lipoprotein receptor-related protein 6; and / or, the truncation of low-density lipoprotein receptor-related protein 5 comprises the intracellular region of the low-density lipoprotein receptor-related protein 5.

68. The modified cell of any one of claims 62-67, wherein the truncation 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 truncation 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 62-68, wherein the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, and SEQ ID NO:

144.

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

71. The modified cell of any one of claims 61-70, which comprises an immune cell.

72. The modified cell of claim 71, wherein the immune cell is selected from the group consisting of a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, and / or a peripheral blood mononuclear cell.

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

74. A method of making the isolated antigen binding protein of any one of claims 1-19, the chimeric antigen receptor of any one of claims 20-56, and / or the polypeptide of claim 57, the method comprising culturing the modified cell of any one of claims 61-73 under conditions such that the isolated antigen binding protein and / or the polypeptide are expressed.

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

76. A method for detecting AFP protein, comprising: Administering the isolated antigen binding protein of any one of claims 1-19, the polypeptide of claim 57, or the immunoconjugate of claim 58.

77. A detection kit for AFP protein, comprising the isolated antigen-binding protein according to any one of claims 1 to 19, the polypeptide according to claim 57, or the immunoconjugate according to claim 58.

78. Use of the isolated antigen-binding protein of any one of claims 1-19, the polypeptide of claim 57, or the immunoconjugate of claim 58 in the preparation of a kit for detecting the presence and / or amount of AFP protein.

79. Use of the isolated antigen-binding protein of any one of claims 1-19, the chimeric antigen receptor of any one of claims 20-56, 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 61-73 in the preparation of a medicament for preventing and / or treating tumors.

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

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

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

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

84. The isolated antigen binding protein of any one of claims 1-19, the chimeric antigen receptor of any one of claims 20-56, 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 61-73, for use in preventing and / or treating tumors.

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

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

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

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

89. 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 of any one of claims 1-19, the chimeric antigen receptor of any one of claims 20-56, 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 61-73.

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

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

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

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

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