ROR1 antigen binding protein and use thereof

Abstract

The present application relates to an isolated antigen-binding protein, which is capable of binding to ROR1. The isolated antigen-binding protein comprises at least one CDR in a variable region of an antibody heavy chain. The present application further provides a pharmaceutical composition comprising the antigen-binding protein, a chimeric antigen receptor, an immunoconjugate, a nucleic acid encoding the antigen-binding protein, a vector containing the nucleic acid molecule, and a cell containing the vector. The present application also provides a use of the antigen-binding protein in preventing, diagnosing, and / or treating a disease.

Description

ROR1 antigen binding protein and its use Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting ROR1. Background Art

[0002] ROR1 is a transmembrane receptor tyrosine kinase protein that consists of an extracellular portion, a transmembrane portion, and a cytoplasmic region. The extracellular region (ECD) contains an immunoglobulin (Ig)-like domain (Ig-Like), a frizzled coiled coil (FZD), and a kringle domain (KD). The intracellular domain (ICD) contains a tyrosine kinase-like domain, two serine / threonine-rich domains, and a proline-rich domain. ROR1 regulates cell division, proliferation, migration, and cell chemotaxis by mediating signal transduction through multiple signaling pathways. ROR1 is highly expressed during early embryonic development and its expression gradually decreases with fetal development. In addition, ROR1 is lowly expressed or not expressed in normal human tissues, but is highly expressed in various hematological malignancies and solid tumors.

[0003] Therefore, ROR1 can serve as a specific tumor marker and a potentially attractive target for tumor treatment. There is an urgent need to develop more therapeutic drugs that can effectively combine with it to meet the treatment needs of a wide range of diseases. Summary of the Invention

[0004] The present application provides an antigen binding protein targeting ROR1. In the present application, the antigen binding protein has one or more of the following properties: (1) it can specifically bind to the ROR1 antigen and has good binding activity; (2) it can specifically bind to ROR1 expressed on the surface of target cells; (3) it can have a killing effect on tumors; (4) it has a certain endocytosis effect and can potentially be used as an antibody targeting portion of an ADC drug. The present application also provides a chimeric antigen receptor comprising the antigen binding protein, and a cell comprising and / or expressing the chimeric antigen receptor, wherein the cell has one or more of the following properties: (1) it can specifically bind to the ROR1 antigen and has good binding activity; (2) it can specifically bind to ROR1 expressed on the surface of target cells; (3) it has good in vitro targeted killing activity; (4) it can secrete more cytokines under the stimulation of target cells; and (5) it has targeted proliferation ability.

[0005] On the one hand, the present application provides an isolated antigen-binding protein that can bind to ROR1, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, the VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132.

[0006] In certain embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 62-92.

[0007] In certain embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NOs:144-155.

[0008] In certain embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-61.

[0009] In certain embodiments, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NOs: 133-143.

[0010] In certain embodiments, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-28.

[0011] In certain embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations:

[0012] (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62;

[0013] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63;

[0014] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 64;

[0015] (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65;

[0016] (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 66;

[0017] (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67;

[0018] (7) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0019] (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 71;

[0020] (9) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0021] (10) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 72;

[0022] (11) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 73;

[0023] (12) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 68;

[0024] (13) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 74;

[0025] (14) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 64;

[0026] (15) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 45, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 75;

[0027] (16) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 69;

[0028] (17) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 76;

[0029] (18) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 77;

[0030] (19) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 78;

[0031] (20) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 70;

[0032] (21) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79;

[0033] (22) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 80;

[0034] (23) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 81;

[0035] (24) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0036] (25) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 52, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 82;

[0037] (26) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83;

[0038] (27) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 84;

[0039] (28) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83;

[0040] (29) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85;

[0041] (30) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 86;

[0042] (31) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 56, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 87;

[0043] (32) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 88;

[0044] (33) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 89;

[0045] (34) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 90;

[0046] (35) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 91; and

[0047] (36) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:92.

[0048] In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4.

[0049] In certain embodiments, the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 93-128.

[0050] In certain embodiments, the antigen binding protein further comprises an immunoglobulin constant region.

[0051] In certain embodiments, the immunoglobulin constant region is an immunoglobulin Fc region.

[0052] In certain embodiments, the immunoglobulin constant region is an IgG Fc region.

[0053] In certain embodiments, the immunoglobulin constant region is a human IgG Fc region.

[0054] In certain embodiments, the antigen binding protein is an antibody or an antigen binding fragment thereof.

[0055] In certain embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

[0056] In certain embodiments, the antigen binding protein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0057] In certain embodiments, the antigen binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0058] In certain embodiments, the antigen binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0059] On the other hand, the present application also provides a chimeric antigen receptor comprising the antigen binding protein.

[0060] In certain embodiments, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0061] In certain embodiments, the antigen binding domain comprises the antigen binding protein.

[0062] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, 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, CD137, CD154 and SLAM.

[0063] In certain embodiments, the transmembrane domain is derived from the transmembrane domain of CD8.

[0064] In certain embodiments, the costimulatory domain comprises a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, 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.

[0065] In certain embodiments, the costimulatory domain is derived from a 4-1BB costimulatory domain.

[0066] In certain embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from any one of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0067] In certain embodiments, the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ.

[0068] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0069] In certain embodiments, the hinge region comprises a hinge region derived from any one of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0070] In certain embodiments, the hinge region is derived from the hinge region of CD8.

[0071] A modified immune cell, wherein the immune cell comprises the chimeric antigen receptor.

[0072] In certain embodiments, the immune cells are T cells, NK cells, iNKT cells, dendritic cells and / or macrophages.

[0073] In certain embodiments, the modified immune cells further comprise and / or express low-density lipoprotein receptor-related protein or a fragment thereof.

[0074] 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.

[0075] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

[0076] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:156.

[0077] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof is an exogenous low-density lipoprotein receptor-related protein or a fragment thereof.

[0078] On the other hand, the present application also provides an immunoconjugate, wherein the immunoconjugate comprises the antigen binding protein.

[0079] In certain embodiments, the immunoconjugate comprises an antigen binding protein, a linker, and a payload.

[0080] In certain embodiments, the payload is a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid.

[0081] In certain embodiments, the immunoconjugate is an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted nuclear drug (RDC).

[0082] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the antigen binding protein and / or the chimeric antigen receptor.

[0083] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0084] On the other hand, the present application also provides a cell comprising the nucleic acid molecule and / or the vector.

[0085] On the other hand, the present application also provides a pharmaceutical composition comprising the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0086] On the other hand, the present application also provides a detection kit, which includes the antigen binding protein, and the detection kit is used to detect the presence and / or content of ROR1 in a sample or a subject.

[0087] On the other hand, the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described in the present application are used in the preparation of a detection kit, and the detection kit is used for diagnosis.

[0088] On the other hand, the present application also provides a method for detecting the presence and / or content of ROR1, which comprises using the antigen binding protein.

[0089] On the other hand, the present application also provides the use of the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing, diagnosing and / or treating a disease and / or condition.

[0090] In certain embodiments, the disease and / or condition is a tumor.

[0091] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0092] In certain embodiments, the tumor is a ROR1-positive tumor.

[0093] In certain embodiments, the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer, or colorectal cancer.

[0094] On the other hand, the present application also provides a method for preventing, diagnosing and / or treating diseases and / or conditions, which comprises administering the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need.

[0095] In certain embodiments, the disease and / or condition is a tumor.

[0096] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0097] In certain embodiments, the tumor is a ROR1-positive tumor.

[0098] In certain embodiments, the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer, or colorectal cancer.

[0099] On the other hand, the present application also provides the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, which are used for preventing, diagnosing and / or treating diseases and / or disorders.

[0100] In certain embodiments, the disease and / or condition is a tumor.

[0101] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0102] In certain embodiments, the tumor is a ROR1-positive tumor.

[0103] In certain embodiments, the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer, or colorectal cancer.

[0104] 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

[0105] 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:

[0106] FIG1 shows the results of POOL ELISA analysis after cell panning described in the present application.

[0107] FIG2 shows the results of POOL ELISA analysis after protein panning described in this application.

[0108] Figures 3A-K show the SEC-HPLC purity test results of the anti-ROR1 antibodies UC961 and 001-010 described in the present application.

[0109] FIG4 shows the analysis results of the flow cytometry binding activity experiment of the anti-ROR1 antibody described in the present application.

[0110] Figure 5 shows a schematic diagram of the various components and connection sequence of the CAR in the ROR1 core plasmid described in this application.

[0111] FIG6 shows the cumulative expansion fold of ROR1CAR-T cells cultured for 13 days after magnetic bead activation as described in the present application.

[0112] FIG7 shows the positive rate results of ROR1CAR-T cells activated by magnetic beads and cultured for 13 days as described in the present application.

[0113] FIG8A shows the results of the cell killing activity of the ROR1CAR-T cells described in the present application against the target cells SKOV3, and FIG8B shows the results of the cell killing activity of the ROR1CAR-T cells described in the present application against the target cells CaoV3.

[0114] Figures 9A-9D show the results of cytokine secretion by ROR1CAR-T cells described in the present application under stimulation of target cells SKOV3 and CaoV3.

[0115] FIG10 shows the results of the targeted proliferation ability of the ROR1CAR-T cells described in the present application.

[0116] FIG11A shows the results of tumor inhibition rate after reinfusion of ROR1CAR-T cells as described in the present application, and FIG11B shows the results of in vivo safety after reinfusion of ROR1CAR-T cells as described in the present application.

[0117] Figures 12A-12B show the results of the endocytic effect of the ROR1 antibody described in this application.

[0118] Figures 13A-13C show the results of the cell killing activity of the ROR1CAR-T cells described in the present application against target cells SKOV3 or MDA-MB-231. DETAILED DESCRIPTION

[0119] 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.

[0120] Definition of terms

[0121] In this application, the terms "ROR1" and "receptor tyrosine kinase orphan receptor 1" are generally used interchangeably and generally refer to a transmembrane receptor tyrosine kinase protein. In this application, the ROR1 can be complete ROR1 and its functionally active fragments, homologs, analogs, variants or derivatives. For example, the ROR1 can be full-length ROR1 or a truncated ROR1 that retains its functional activity. In this application, the ROR1 can be of any species. For example, the ROR1 can be human ROR1. In this application, the ROR1 can be artificially synthesized ROR1. In this application, the ROR1 can be wild-type or artificially modified. For example, the ROR1 can be modified ROR1.

[0122] In this application, the term "antigen binding protein" generally refers to a protein with the ability to bind to an antigen. In this application, the antigen binding protein may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the antigen binding portion to bind to the antigen. In this application, the antigen binding protein may be an artificially synthesized antigen binding protein. In this application, the antigen binding protein may be wild-type or artificially modified. In this application, the antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with transplanted CDRs or CDR derivatives. In this application, the antigen binding protein may be an antibody or an antigen-binding fragment thereof, as well as variants, homologues, derivatives or analogs thereof. In the present application, antigen-binding proteins may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, as long as they exhibit the desired antigen-binding activity. For example, the antigen-binding protein may be a VHH.

[0123] In this application, the term "VHH" generally refers to an antibody structure consisting of a heavy chain variable region. In this application, the VHH may comprise a complementarity determining region (CDR) and a framework region (FR). In this application, the VHH comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In this application, the VHH may be wild type or artificially synthesized. In this application, the VHH may be of animal or non-animal origin. In this application, the VHH may be artificially modified. In this application, the VHH may be a chimeric antibody, a humanized antibody, or a fully human antibody.

[0124] In this application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably, and the constant region refers to the region of the non-variable region of an antibody. In this application, the constant region is not directly involved in binding to an antigen, but exhibits various effector functions. In this application, the constant region can be the entire non-variable region of an antibody, or it can be a portion of the non-variable region of an antibody. For example, the constant region can be a heavy chain constant region. For example, the constant region can be an Fc region. In this application, the constant region can be derived from an animal. For example, the constant region can be derived from humans, goats, rabbits, rats, or guinea pigs. For example, the Fc region can be a human Fc region.

[0125] The proteins and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the amino acid sequence of the protein (e.g., the antigen-binding protein described in this application). For example, the functional variant may include a protein or polypeptide that has been subjected to amino acid changes by at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution can be a conservative substitution.

[0126] In the present application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, the variable region and constant region of an antibody may both be derived from the variable region and constant region of an antibody from a single animal species (e.g., human).

[0127] In the present application, the homolog can be a protein or polypeptide 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 homology with the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen-binding protein described herein).

[0128] In this application, the terms "chimeric antigen receptor" and "CAR" are generally used interchangeably and generally refer to a recombinant polypeptide that comprises at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. In this application, CAR may sequentially include an optional signal peptide, an antigen binding domain, a hinge region, a transmembrane domain region, and an intracellular signaling domain. For example, the CAR may also include an optional intracellular costimulatory domain. In this application, the antigen binding domain may be wild-type or artificially modified. In this application, the antigen binding domain may be artificially synthesized. In this application, the antigen binding domain may be an antigen binding protein. In this application, the antigen binding protein may be an antibody or an antigen binding fragment thereof. For example, the antigen binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment. In the present application, the chimeric antigen receptor may also include low-density lipoprotein receptor-related protein or a fragment thereof.

[0129] In the present application, the term "intracellular domain" generally refers to an intracellular domain comprising any truncated portion sufficient to transduce an activation signal. The intracellular domain may include intracellular signals and / or costimulatory domains. The term "intracellular signaling domain" refers to an intracellular region that can generate a signal that promotes the immune effector function of CAR cells (e.g., CART cells or NK cells expressing CAR). For example, the intracellular signaling region may include an intracellular signaling region of one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and at least one domain comprising an ITAM. For example, the intracellular signaling region may be a signaling domain derived from CD3ζ. The term "costimulatory domain" refers to a part of the CAR that can transduce effector signals in the intracellular signal region. For example, the costimulatory domain may include an intracellular costimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, 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 domain may be an intracellular costimulatory domain derived from 4-1BB.

[0130] In this application, the term "transmembrane domain" generally refers to a domain of a peptide, polypeptide or protein that is capable of crossing the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane. For example, the membrane-spanning region can include the membrane-spanning domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, 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, CD137, CD154, and SLAM. For example, the membrane-spanning domain can be derived from the membrane-spanning domain of CD8.

[0131] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein that is an endocytic receptor. It is widely distributed in the body and has great differences between tissues. Its main function is to take up cholesterol into cells for cell proliferation and the synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein can be from any vertebrate. For example, the low-density lipoprotein receptor-related protein or its fragment can be located at the C-terminus of the intracellular signaling region. For example, the low-density lipoprotein receptor-related protein or its fragment can include one or more selected from the following group: low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or its fragment can be low-density lipoprotein receptor-related protein 6 or its fragment.

[0132] In this application, the term "immune cell" generally refers to a cell that participates in an immune response. For example, the immune cell can be an immune cell that performs effector functions. For example, the effector functions can include removing foreign antigens or promoting immune effector responses, etc. In this application, the immune effector cell can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells and / or pluripotent stem cells. For example, the immune effector cell can be a T cell.

[0133] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by the conjugation of other active ingredients with the antigen-binding proteins. In the present application, the active ingredient can be covalently linked to the antigen-binding proteins by a connecting molecule. In the present application, the immunoconjugate can be a conjugation of an antigen-binding protein with a payload. For example, the payload can be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid. In the present application, the immunoconjugate can be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted nuclear medicine (RDC). In the present application, the conjugate can be specifically bound to the antigen on the target cell by the antigen-binding protein, and the payload is delivered to the target cell. For example, the target cell can be a tumor cell.

[0134] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length, either deoxyribonucleotides or ribonucleotides, in isolated form, or analogs thereof isolated from their natural environment or synthesized artificially. For example, the nucleic acid molecule can be a DNA molecule or an RNA molecule. For example, the nucleic acid molecule can be single-stranded or double-stranded. For example, the nucleic acid molecule can encode an antigen-binding protein and / or a chimeric antigen receptor.

[0135] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in the host cells. For example, the vector may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0136] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. A cell can include the offspring of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). A cell can include a cell transfected in vitro with a vector as described herein. The cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, a human non-small cell lung cancer A549 cell, a human skin squamous cell carcinoma A431 cell, a renal clear cell adenocarcinoma 786-0 cell, a human pancreatic cancer MIAPaCa-2 cell, an erythroleukemia K562 cell, an acute T-cell leukemia Jurkat cell, a human breast cancer MCF-7 cell, a human breast cancer MDA-MB-231 cell, a human breast cancer MDA-MB-468 cell, a human breast cancer SKBR3 cell, a human ovarian cancer SKOV3 cell, a lymphoma U-937 cell, a lymphoma Raji cell, a human myeloma U266 cell, or a human multiple myeloma RPMI8226 cell. In certain embodiments, the cell is a mammalian cell.

[0137] As used herein, the term "pharmaceutically acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dose and concentration employed. For example, a physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0138] In this application, the term "prevention, diagnosis and / or treatment" generally refers to the prevention, diagnosis and / or treatment of a disease. For example, the prevention, diagnosis and / or treatment may be preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, preventing further increase in the severity of the disease and any symptoms associated therewith, or making a judgment on a physiological or mental disease of the human body and its pathological causes. In this application, the disease may be a tumor disease. For example, preventing, diagnosing or alleviating the onset of one or more symptoms associated with a tumor, reducing the severity and duration of a tumor and symptoms associated therewith.

[0139] In the present application, the term "tumor" generally refers to any new pathological tissue proliferation in the presence of tumor antigens that can be recognized by the immune system. In the present application, the tumor may include benign or malignant tumors (cancer). In the present application, the cancer may be metastatic cancer and non-metastatic cancer. In the present application, the tumor may include solid tumors and hematologic tumors. In the present application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor may include a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the hematologic tumor generally refers to a class of hematopoietic system diseases. In the present application, the hematologic tumor may include various types of leukemia, multiple myeloma or malignant lymphoma. In the present application, the tumor may be a tumor that is positive for ROR1 expression. For example, the tumor can be chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer, or colorectal cancer.

[0140] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0141] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0142] 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.

[0143] Detailed Description of the Invention

[0144] Antigen binding proteins

[0145] 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, and a combination of Kabat / Chothia. 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 homology to the amino acid sequence of the CDR. For example, the CDRs of the isolated antigen-binding proteins described in the sequence listing of the present application can be determined using Kabat.

[0146] In one aspect, the present application provides an isolated antigen-binding protein, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, and the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and / or HCDR3.

[0147] In the present application, the antigen-binding protein has one or more of the following properties: (1) the ability to specifically bind to the ROR1 antigen and have good binding activity; (2) the ability to specifically bind to ROR1 expressed on the surface of target cells; (3) the ability to kill tumor cells; (4) a certain endocytosis effect, and the potential to be used as the antibody targeting portion of an ADC drug. For example, the antigen-binding protein can bind to ROR1 on the surface of target cells with an affinity / binding activity higher than that of the control antibody UC961. For example, the isolated antigen-binding protein can bind to the ROR1 protein with a lower KD than that of the control antibody. For example, the antigen-binding protein can have an endocytosis effect that is better than that of the control antibody UC961. For example, the antigen-binding protein can have an endocytosis effect comparable to that of the control antibody UC961.

[0148] In the present application, the KD value can also be determined by ELISA, competitive ELISA, BIACORE, or KINEXA. In the present application, the isolated antigen-binding protein can specifically bind to human ROR1 protein. The specific binding can be determined by FACS.

[0149] In the present application, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 62, SEQ ID NOs: 65-70, SEQ ID NOs: 72-77, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NOs: 85-92 and SEQ ID NOs: 129-132.

[0150] In the present application, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 62-92.

[0151] In the present application, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 33, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NOs: 144-155.

[0152] In the present application, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 29-61.

[0153] In the present application, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 24 and SEQ ID NOs: 133-143.

[0154] In the present application, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-28.

[0155] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NOs:144-155, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NOs:133-143.

[0156] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NOs:144-155, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:1-28.

[0157] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:29-61, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NOs:133-143.

[0158] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:62-92, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NOs:144-155, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NOs:133-143.

[0159] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:29-61, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:1-28.

[0160] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:62-92, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NOs:144-155, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs:1-28.

[0161] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs:62-92, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs:29-61, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:24 and SEQ ID NOs:133-143.

[0162] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 62-92, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-61, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-28.

[0163] In the present application, the amino acid sequences of HCDR1, HCDR2 and HCDR3 may be selected from any one of the following combinations:

[0164] (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 144, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62;

[0165] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 139, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 145, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 129;

[0166] (3) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 134, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 130;

[0167] (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 147, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65;

[0168] (5) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 133, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 66;

[0169] (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 145, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67;

[0170] (7) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 148, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0171] (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 133, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 150, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 130;

[0172] (9) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 135, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0173] (10) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 134, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 72;

[0174] (11) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 152, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 73;

[0175] (12) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 133, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 149, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 68;

[0176] (13) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 136, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 148, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 74;

[0177] (14) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 138, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 130;

[0178] (15) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 135, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 147, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 75;

[0179] (16) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 147, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 69;

[0180] (17) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 138, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 76;

[0181] (18) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 140, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 77;

[0182] (19) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 133, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 149, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 132;

[0183] (20) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 140, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 144, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 70;

[0184] (21) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 141, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 146, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 132;

[0185] (22) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 150, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 131;

[0186] (23) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 136, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 131;

[0187] (24) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 138, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 154, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0188] (25) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 142, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 153, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 82;

[0189] (26) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 144, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 129;

[0190] (27) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 139, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 152, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 84;

[0191] (28) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 144, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 129;

[0192] (29) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 155, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85;

[0193] (30) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 137, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 152, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 86;

[0194] (31) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 141, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 155, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 87;

[0195] (32) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 154, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 88;

[0196] (33) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 143, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 89;

[0197] (34) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 142, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 145, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 90;

[0198] (35) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 135, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 91; and

[0199] (36) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 143, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 149, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 92.

[0200] In the present application, the amino acid sequences of HCDR1, HCDR2 and HCDR3 may be selected from any one of the following combinations:

[0201] (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62;

[0202] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63;

[0203] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 64;

[0204] (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65;

[0205] (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 66;

[0206] (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67;

[0207] (7) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0208] (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 71;

[0209] (9) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0210] (10) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 72;

[0211] (11) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 73;

[0212] (12) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 68;

[0213] (13) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 74;

[0214] (14) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 64;

[0215] (15) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 45, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 75;

[0216] (16) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 69;

[0217] (17) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 76;

[0218] (18) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 77;

[0219] (19) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 78;

[0220] (20) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 70;

[0221] (21) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79;

[0222] (22) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 80;

[0223] (23) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 81;

[0224] (24) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67;

[0225] (25) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 52, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 82;

[0226] (26) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83;

[0227] (27) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 84;

[0228] (28) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83;

[0229] (29) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85;

[0230] (30) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 86;

[0231] (31) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 56, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 87;

[0232] (32) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 88;

[0233] (33) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 89;

[0234] (34) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 90;

[0235] (35) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 91; and

[0236] (36) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:92.

[0237] In the present application, AAAX1GX2RX3EDGRVRTLPSX4YX5X6 (SEQ ID NO: 129), wherein X1 can be R or a deletion of an amino acid; X2 can be L or V; X3 can be A or Y; X4 can be D or E; X5 can be T or a deletion of an amino acid; and X6 can be F or a deletion of an amino acid.

[0238] In the present application, AAX1RX2X3SRDX4YNYDY (SEQ ID NO: 130), wherein X1 can be R or V; X2 can be F or I; X3 can be R or a deletion of an amino acid; and X4 can be R or a deletion of an amino acid.

[0239] In the present application, AAGDYGX1X2SX3DYNY (SEQ ID NO: 131), wherein X1 can be N or S; X2 can be F or Y; and X3 can be S or Y.

[0240] In the present application, AARRFX1X2X3X4X5YX6Y (SEQ ID NO: 132), wherein X1 can be N or T; X2 can be G or a deletion of an amino acid; X3 can be R or S; X4 can be W or Y; X5 can be S or T; and X6 can be D or N.

[0241] In the present application, GX1X2FSIDR (SEQ ID NO: 133), wherein X1 can be R or D; X2 can be I or T.

[0242] In the present application, GRIFSPDX1 (SEQ ID NO: 134), wherein X1 can be I or S.

[0243] In the present application, GRTX1SSX2A (SEQ ID NO: 135), wherein X1 can be F or L; X2 can be N or Y.

[0244] In the present application, GFIFSANX1 (SEQ ID NO: 136), wherein X1 can be A or P.

[0245] In the present application, GX1IFSX2YX3 (SEQ ID NO: 137), wherein X1 can be F or N; X2 can be L or T; and X3 can be R or T.

[0246] In the present application, GX1X2LSX3NA (SEQ ID NO: 138), wherein X1 can be G, R or Y; X2 can be I or T; and X3 can be D, I or Y.

[0247] In the present application, GX1TFSRX2X3 (SEQ ID NO: 139), wherein X1 can be R or Y; X2 can be D or N; and X3 can be I or P.

[0248] In the present application, GX1TFSX2YX3 (SEQ ID NO: 140), wherein X1 can be R or S; X2 can be A or I; and X3 can be I or T.

[0249] In the present application, GYIX1SX2YX3 (SEQ ID NO: 141), wherein X1 can be F or L; X2 can be T or Y; and X3 can be D or Y.

[0250] In the present application, GLTFX1X2X3A (SEQ ID NO: 142), wherein X1 can be R or S; X2 can be L or Y; and X3 can be N or Y.

[0251] In the present application, GRSFX1X2SX3 (SEQ ID NO: 143), wherein X1 can be R or S; X2 can be D or S; and X3 can be A or I.

[0252] In the present application, ISRX1GGX2T (SEQ ID NO: 144), wherein X1 can be R or T; X2 can be I or S.

[0253] In the present application, INX1RX2GSX3T (SEQ ID NO: 145), wherein X1 can be S or a deletion of an amino acid; X2 can be D, V or a deletion of an amino acid; and X3 can be G, S or a deletion of an amino acid.

[0254] In the present application, ISWGDSX1T (SEQ ID NO: 146), wherein X1 can be N or V.

[0255] In the present application, ITSX1GGX2T (SEQ ID NO: 147), wherein X1 can be G, R or S; X2 can be L or Y.

[0256] In the present application, INDGX1GX2T (SEQ ID NO: 148), wherein X1 can be D or G; X2 can be R or Y.

[0257] In the present application, ISX1SGX2GX3T (SEQ ID NO: 149), wherein X1 can be S, T or a deletion of an amino acid; X2 can be D, S or a deletion of an amino acid; and X3 can be L, T or a deletion of an amino acid.

[0258] In the present application, ISX1GX2SX3T (SEQ ID NO: 150), wherein X1 can be R or G; X2 can be D or G; and X3 can be I or T.

[0259] In the present application, ITX1SDGX2T (SEQ ID NO: 151), wherein X1 can be R or S; X2 can be S or V.

[0260] In the present application, IX1WX2GGX3T (SEQ ID NO: 152), wherein X1 can be N, S or T; X2 can be G, R or S; and X3 can be N, R or T.

[0261] In the present application, INTX1X2GX3T (SEQ ID NO: 153), wherein X1 can be G or S; X2 can be D or G; and X3 can be L or S.

[0262] In the present application, ITTGX1GX2T (SEQ ID NO: 154), wherein X1 can be G or S; X2 can be G or L.

[0263] In the present application, ISAX1GX2X3T (SEQ ID NO: 155), wherein X1 can be R or S; X2 can be G or S; and X3 can be V or Y.

[0264] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and / or H-FR4.

[0265] In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 may be located between the HCDR1 and the HCDR2, the H-FR3 may be located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 may be directly or indirectly connected to the C-terminus of the HCDR3.

[0266] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and / or H-FR4, and the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0267] In the present application, the amino acid sequence of the FR may be FR of any species, for example, the FR may be FR of mouse, rabbit, goat, alpaca or human origin.

[0268] In the present application, the amino acid sequence of FR can be adjusted as needed. For example, the FR can be a wild-type sequence. In the present application, the FR can be an artificially modified sequence. For example, during the process of antibody humanization, the amino acid sequence of FR can be changed without reducing the binding activity / affinity of the antigen binding protein to ROR1. For example, during the process of antibody humanization, one or more amino acid sequences in FR can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to ROR1. For example, the FR can be a variant thereof, and the variant includes the amino acid sequence of the FR being substituted, deleted and / or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (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 to the amino acid sequence of the FR.

[0269] In the present application, the direct or indirect connection may be connected through intermolecular forces, or may be connected through a linker.

[0270] In the present application, the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs:93-128. For example, the VH may be a wild-type sequence. For example, the VH may be an artificially engineered sequence. For example, one or more amino acid sequences in the VH may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to ROR1. For example, the VH may be a variant thereof, wherein the variant comprises a substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence of the FR. For example, the VH may also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in any one of SEQ ID NOs:93-128.

[0271] In the present application, the antigen binding protein may further comprise an immunoglobulin constant region. For example, the immunoglobulin constant region may be an antibody heavy chain constant region.

[0272] In the present application, the immunoglobulin constant region may be an immunoglobulin Fc region. For example, the Fc region may be a wild-type sequence, or may be mutated or optimized. For example, the Fc region may be an artificially engineered sequence. For example, the immunoglobulin constant region may be an IgG Fc region. For example, the immunoglobulin constant region may be an IgG1 Fc region. For example, the immunoglobulin constant region may be an IgG4 Fc region. For example, the Fc region may be derived from a mouse, rabbit, goat, alpaca, or human Fc region. For example, the immunoglobulin constant region may be a human IgG Fc region.

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

[0274] In the present application, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

[0275] In the present application, the antigen-binding protein can be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen-binding protein can be mutated / optimized in the FR and constant regions without reducing the binding activity / affinity of the antigen-binding protein to ROR1. For example, the antigen-binding protein can have low immunogenicity without reducing the binding activity / affinity of the antigen-binding protein to ROR1.

[0276] In the present application, the antigen-binding protein can be a monospecific antibody, a bispecific antibody, or a multispecific antibody. For example, the antigen-binding protein can further comprise a second domain. For example, the second domain can target ROR1 or another antigen. For example, the second domain can target a hematologic malignancy / solid tumor antigen. For example, the antigen-binding protein can further comprise a third domain. For example, the third domain can target ROR1 or another antigen. For example, the third domain can target a hematologic malignancy / solid tumor antigen.

[0277] In the present application, the antigen-binding protein can be a monovalent antibody, a bivalent antibody, or a multivalent antibody. For example, the antigen-binding protein has one antigen-binding site. For example, the antigen-binding protein has two antigen-binding sites. For example, the antigen-binding protein has multiple antigen-binding sites.

[0278] In the present application, the antigen-binding protein can be expressed as a fusion protein. For example, the antigen-binding protein can be fused to a peptide, polypeptide, amino acid, or protein. For example, the antigen-binding protein can be fused to one or more functional molecules. For example, the functional molecules can be one or more antigen-binding proteins. For example, the functional molecules can be one or more Fc regions.

[0279] In the present application, the antigen-binding protein may be wild-type. For example, the antigen-binding protein may comprise a wild-type sequence. In the present application, the antigen-binding protein may be an artificially engineered antigen-binding protein. In the present application, the antigen-binding protein may be recombinant. For example, the antigen-binding protein may be mutated or optimized.

[0280] Antigen recognition receptors

[0281] On the other hand, the present application provides an antigen recognition receptor comprising the antigen binding protein.

[0282] In the present application, the antigen recognition receptor may be a chimeric antigen receptor (CAR). In the present application, the antigen recognition receptor may be a T cell receptor (TCR).

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

[0284] In the present application, the antigen binding domain may comprise the antigen binding protein.

[0285] For example, the antigen binding domain can be an antibody or an antigen binding fragment thereof.

[0286] In the present application, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment.

[0287] For example, the antigen binding domain can comprise an antigen binding protein comprising an antibody heavy chain variable region VH, wherein the VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 or SEQ ID NOs:129-132; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:60 or SEQ ID NOs:144-155; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:24 or SEQ ID NOs:133-143.

[0288] For example, the antigen-binding domain may comprise an antigen-binding protein, which comprises an antibody heavy chain variable region VH, wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NOs: 62-92; the HCDR2 comprises the amino acid sequence shown in SEQ ID NOs: 29-61; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NOs: 1-28.

[0289] For example, the antigen-binding domain may comprise an antigen-binding protein comprising an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NOs: 93-128.

[0290] In the present application, the antigen binding domain of the chimeric antigen receptor may comprise CDR1, CDR2 and CDR3.

[0291] In the present application, the transmembrane domain may comprise a transmembrane domain derived from the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, 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, CD137, CD154 or SLAM.

[0292] In the present application, the transmembrane domain may be derived from the transmembrane domain of CD8.

[0293] In the present application, the costimulatory domain may comprise a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, 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.

[0294] In the present application, the costimulatory domain may be derived from a 4-1BB costimulatory domain.

[0295] In the present application, the intracellular signaling domain may comprise an intracellular signaling domain derived from the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 or a domain comprising at least one ITAM.

[0296] In the present application, the intracellular signaling domain may be derived from the intracellular signaling domain of CD3ζ.

[0297] In the present application, the chimeric antigen receptor may further comprise a hinge region.

[0298] In the present application, the hinge region may comprise a hinge region derived from the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

[0299] In the present application, the hinge region may be derived from the hinge region of CD8.

[0300] In the present application, the CAR may further comprise a low-density lipoprotein receptor-related protein or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may include low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof. In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 156. In the present application, the low-density lipoprotein receptor-related protein or a fragment thereof may be an exogenous low-density lipoprotein receptor-related protein or a fragment thereof.

[0301] In the present application, the sequence of the low-density lipoprotein receptor-related protein or its fragment in the CAR can be connected to the C-terminal sequence of CAR by a self-cleavage peptide (e.g., 2A peptides such as T2A, P2A, and E2A). For example, the low-density lipoprotein receptor-related protein or its fragment can be connected to the C-terminal of the intracellular signaling region by T2A. In the present application, the chimeric antigen receptor may include an antigen binding domain targeting ROR1, a CD8 transmembrane domain, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain.

[0302] In the present application, the chimeric antigen receptor may comprise an antigen binding domain targeting ROR1, a hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain.

[0303] In the present application, the chimeric antigen receptor may comprise a signal peptide, an antigen binding domain targeting ROR1, a hinge region, a transmembrane domain, an intracellular costimulatory domain and an intracellular signaling domain.

[0304] In the present application, the chimeric antigen receptor may comprise a signal peptide, an antigen binding domain targeting ROR1, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, an intracellular signaling domain and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 156.

[0305] In the present application, the chimeric antigen receptor may comprise an antigen binding protein targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0306] In the present application, the chimeric antigen receptor may comprise a signal peptide, an antigen binding protein targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0307] In the present application, the chimeric antigen receptor may comprise a signal peptide, an antigen binding protein targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, a CD3ζ intracellular signaling domain, and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 156.

[0308] In the present application, the chimeric antigen receptor may comprise a VHH targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0309] In the present application, the chimeric antigen receptor may comprise a signal peptide, a VHH targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0310] In the present application, the chimeric antigen receptor may comprise a signal peptide, a VHH targeting ROR1, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain, a CD3ζ intracellular signaling domain and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 156.

[0311] In the present application, the TCR may comprise the antigen binding protein.

[0312] In the present application, the TCR can specifically bind to ROR1.

[0313] In the present application, the TCR may comprise an α chain and a β chain, wherein the α chain comprises an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprises a β chain variable region (TRBV) and a β chain constant region (TRBC).

[0314] For example, the TRAV and / or TRBV may comprise three hypervariable regions CDR1, CDR2 and CDR3.

[0315] On the other hand, the present application provides a modified immune cell, wherein the immune cell may comprise the chimeric antigen receptor and / or T cell receptor.

[0316] In the present application, the immune cells can be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, γδT cells and / or DNT cells. For example, the immune cells can be immune effector cells. For example, the immune cells can be T cells. For example, the immune cells can be a mixture, and the mixture can contain different types of immune cells, for example, the mixture can contain one or more immune cells.

[0317] In the present application, the immune cells may contain and / or express one or more antigen recognition receptors. In the present application, the immune cells may contain and / or express one or more antigen recognition receptors.

[0318] In the present application, the immune cells may comprise and / or express one or more chimeric antigen receptors. In the present application, the immune cells may comprise and / or express one or more chimeric antigen receptors.

[0319] In the present application, the cell may contain and / or express the CAR and the low-density lipoprotein receptor-related protein or a fragment thereof.

[0320] Immunoconjugates

[0321] On the other hand, the present application also provides an immunoconjugate, wherein the immunoconjugate may comprise the antigen binding protein. In the present application, the immunoconjugate may specifically bind to ROR1.

[0322] In the present application, the immunoconjugate may comprise an antigen binding protein, a linker, and a payload. In the present application, the parts of the immunoconjugate are directly or indirectly connected. For example, the parts of the immunoconjugate are indirectly connected. For example, the parts of the immunoconjugate are connected via a linker.

[0323] In the present application, the payload may be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound or a glucocorticoid.

[0324] In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC) or a targeted nuclear drug (RDC).

[0325] In the present application, the antibody drug conjugate may comprise the antigen binding protein, a linker, and a cytotoxic drug. In the present application, the cytotoxic drug may be a chemotherapeutic agent, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In the present application, the chemotherapeutic agent may be vinblastine or doxorubicin. In the present application, the tubulin inhibitor may be auristatin, eribulin, or maytansine. In the present application, the DNA damaging agent may be calicheamicin, duocarmycin, or anthramycin derivative PBD. In the present application, the topoisomerase I inhibitor may be camptothecin, hydroxycamptothecin, irinotecan, or topotecan.

[0326] In the present application, the PROTAC-antibody conjugate may comprise the antigen binding protein, a linker, and a PROTAC. In the present application, the PROTAC may comprise a target protein ligand, a linker, and an E3 ligase ligand. In the present application, the E3 ligase ligand may be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0327] In the present application, the targeted nuclear medicine may comprise the antigen binding protein, a linker and a radioactive isotope. In the present application, the radioactive isotope may be iodine-131, radium-223, thallium-201 or arsenic-211.

[0328] Nucleic acid molecules, vectors and cells

[0329] On the other hand, the present application also provides an isolated nucleic acid molecule, which can encode the antigen binding protein and / or the chimeric antigen receptor.

[0330] In the present application, the nucleic acid molecule may further comprise a sequence encoding a signal peptide.

[0331] In the present application, the nucleic acid molecule may further comprise a sequence encoding the low-density lipoprotein receptor-related protein or a fragment thereof. For example, the nucleic acid molecule comprises a sequence capable of expressing the chimeric antigen receptor and the low-density lipoprotein receptor-related protein or a fragment thereof in a cell. In the present application, the nucleic acid sequence encoding the chimeric antigen receptor may be linked to the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or a fragment thereof via a cleavage peptide. For example, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof may comprise the nucleotide sequence shown in SEQ ID NO: 157.

[0332] In the present application, the nucleic acid molecule can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cleavage and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.

[0333] In the present application, the nucleic acid molecule may be DNA and / or RNA.In the present application, the nucleic acid molecule may be an artificially synthesized nucleic acid analog.

[0334] In the present application, the nucleic acid molecule may be a modified nucleic acid molecule.

[0335] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0336] In the present application, the vector may comprise one or more nucleic acid molecules. In the present application, the vector may comprise one or more nucleic acid molecules.

[0337] In the present application, the vector may be an expression vector or a cloning vector. In the present application, the vector may be a viral vector or a non-viral vector. In the present application, the vector may be a viral vector, a plasmid vector, a phage vector or other vectors commonly used in, for example, genetic engineering. For example, the viral vector may be an adenovirus, an adeno-associated virus, a retrovirus (including a lentivirus). In the present application, the vector may be a fusion vector or a non-fusion vector.

[0338] In the present application, the vector may further comprise other genes. For example, the other genes may be marker genes.

[0339] In the present application, the vector may contain a variety of elements that control expression. For example, the vector may include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector may also contain a replication initiation site. For example, the vector may include components that assist entry into cells. In order to allow the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.

[0340] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0341] In the present application, the cell may include the progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny may not necessarily be completely identical to the original parent cell (in the form of total DNA complement or in the genome).

[0342] In the present application, the cell can be a prokaryotic cell (eg, a bacterial cell), a CHO cell, a NS / 0 cell, a HEK293T cell or a HEK293A cell, or other eukaryotic cells, such as a fungal or yeast cell.

[0343] In the present application, the cell may comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector may comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0344] In the present application, the vector can be introduced into the cell by methods known in the art. For example, the method can be electroporation, lipofectine transfection or lipofectamin transfection.

[0345] On the other hand, the present application provides a method for preparing the antigen-binding protein, which comprises culturing the cells under conditions allowing the antigen-binding protein to be expressed.

[0346] Pharmaceutical composition

[0347] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0348] In the present application, the pharmaceutical composition can include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present application can include liquid, frozen and lyophilized compositions.

[0349] In this application, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0350] In the present application, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0351] Drug combinations

[0352] On the other hand, the present application provides a pharmaceutical combination comprising the antigen-binding protein. The pharmaceutical combination may further comprise one or more active ingredients other than the antigen-binding protein.

[0353] For example, the drug combination may further comprise a substance associated with an immune response. For example, the drug combination may further comprise a drug associated with an immune response.

[0354] On the other hand, the present application provides a pharmaceutical combination comprising the antigen binding protein, the nucleic acid molecule and / or the cell and a therapeutic agent. In the present application, the therapeutic agent can be selected from one or more of the following groups: an anti-tumor drug, a chemotherapeutic agent, a radioisotope or an immune checkpoint inhibitor.

[0355] On the other hand, the present application also provides a scheme for combining the antigen-binding protein with one or more other active ingredients. For example, the antigen-binding protein is combined with one or more other therapeutic agents. In the present application, the drug combination can be administered separately, simultaneously, or sequentially. In the present application, the drug combination can be administered in the same or different doses or administration routes. For example, the active ingredients in the drug combination are administered to the patient as separate entities in the same / different doses and administration routes. In the present application, the ingredients in the drug combination can be administered to the patient simultaneously in the form of a single entity or dose. For example, the ingredients in the drug combination are administered to the patient simultaneously, jointly, or sequentially as separate entities. In the present application, the specific administration route can be determined according to the type of active ingredient, and the specific dosage can be adjusted according to the severity of the subject's condition, the subject's physical condition, etc.

[0356] In the present application, the different active ingredients in the pharmaceutical combination may be mixed or placed separately. For example, the active ingredients may be placed in the same container. For example, the active ingredients may be placed in different containers.

[0357] Preparation method

[0358] In another aspect, the present application provides a method for preparing the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition. For example, the method may include culturing the cell under conditions that allow the antigen-binding protein and / or the chimeric antigen receptor to be expressed. For example, the method may include introducing the vector into the immune cell.

[0359] use

[0360] On the other hand, the present application also provides the use of the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug, which can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0361] On the other hand, the present application also provides a method for preventing, diagnosing and / or treating diseases and / or conditions, which may include administering the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need.

[0362] On the other hand, the present application also provides the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, which can be used for preventing, diagnosing and / or treating diseases and / or disorders.

[0363] In the present application, the disease and / or disorder may be a ROR1-related disease and / or disorder.

[0364] In the present application, the disease and / or condition can be a tumor. In the present application, the tumor can be a solid tumor and / or a hematologic tumor. In the present application, the tumor can be a ROR1-positive tumor. In the present application, the hematologic tumor can be chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL). In the present application, the solid tumor can be ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, renal cancer, liver cancer, thyroid cancer or colorectal cancer.

[0365] In the present application, the prevention, diagnosis and / or treatment may be preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0366] On the other hand, the present application provides use of the antigen binding protein for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose diseases and / or disorders related to the expression of the ROR1 protein.

[0367] On the other hand, the present application provides use of the antigen binding protein for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose diseases and / or disorders related to the expression of the ROR1 protein.

[0368] In another aspect, the present application provides the antigen binding protein for use in diagnosing diseases and / or disorders associated with the expression of ROR1 protein.

[0369] In the present application, the diagnostic agent can be used alone or in combination with an instrument, apparatus, device, or system. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of genetic diseases, the diagnostic agent can be used to perform in vitro detection on human samples (e.g., various body fluids, cells, tissue samples, etc.).

[0370] In the present application, the diagnostic agent can be selected from the following groups: reagents, kits, calibrators and quality control products.

[0371] In the present application, the in vitro detection method can be selected from the following group: Western Blot, ELISA and immunohistochemistry.

[0372] For example, the reagent may include a reagent capable of measuring the expression amount of the ROR1 protein.

[0373] For example, the reagent may be selected from the following group: a reagent for performing Western Blot, a reagent for performing ELISA, and a reagent for performing immunohistochemistry.

[0374] On the other hand, the present application also provides a detection kit, which may include the antigen binding protein, and the detection kit is used to detect the presence and / or content of ROR1 in a sample or subject. For example, the detection kit can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0375] For example, the present application relates to an immunoassay kit using the antibodies of the present application in combination with immunoassay methods such as ELISA, immunohistochemistry, protein blotting, and flow cytometry.

[0376] In the present application, the kit includes the antigen binding protein of the present application for detecting ROR1-related cancer cells. Specifically, the immunoassay kit will contain the antibody of the present application as the first antibody binding to ROR1, and optional immunoassay reagents in a suitable container component.

[0377] For example, the antibody can be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well.

[0378] In the present application, the immunodetection reagents in the kit can be in any of a variety of forms, including those detectable labels that are bound or linked to a given antibody. Detectable labels that are bound or linked to a secondary binding ligand can also be included. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0379] In the present application, other immunoassay reagents suitable for use in the kits of the present application include a two-component reagent comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As described above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present application.

[0380] On the other hand, the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described in the present application are used in the preparation of a detection kit, and the detection kit is used for diagnosis.

[0381] In the present application, the kit may further comprise an appropriate aliquot of a ROR1 composition, whether labeled or unlabeled, which can be used to prepare a standard curve for detection analysis. The kit may contain the antibody-label conjugate in a fully conjugated form, in an intermediate form, or in separate portions that are conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in a lyophilized form.

[0382] In the present application, the container means of the test kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody or preferably an appropriate aliquot of the antibody can be placed. The test kit of the present application will also typically include means for containing the antibody, antigen, and any other reagent containers, which are in a sealed form for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the vials are retained.

[0383] On the other hand, the present application also provides a method for detecting the presence and / or content of ROR1, which may include using the antigen binding protein.

[0384] On the other hand, the present application provides a method for diagnosing a disease and / or condition related to the expression of ROR1 protein in a subject, the method comprising: contacting a sample derived from the subject with the antigen binding protein, and determining the presence and / or content of a substance in the sample that can specifically bind to the antigen binding protein.

[0385] In another aspect, the present application provides a method for detecting ROR1 in a sample or subject, comprising administering the antigen-binding protein. In the present application, the administration can be performed in various ways, such as intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0386] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0387] Example

[0388] Example 1 Screening of anti-ROR1 antibodies using the artificially synthesized nanoantibody library NanoOri1.0

[0389] By comparing 296 non-repetitive sequence nanobodies and their structures in the PDB database (Protein Data Bank), the CDRs mutation strategy was determined, among which the lengths of CDR3 were three types: 14, 17 and 21 amino acids. The already marketed nanobody Caplacizumab was selected as the backbone, and the nucleotide sequence of Caplacizumab was fully synthesized. It was then cloned into the HP153 phage vector, and then the single-stranded DNA of HP153 was extracted. The CDRs of the nanobody were mutated using the Kunkel mutagenesis method to obtain double-stranded DNA. The double-stranded DNA was electroporated into competent Escherichia coli SS320 pre-infected with M13KO7 helper phage. After overnight culture, the phage supernatant was collected, and finally a diversity of 1.44×10 10 The artificial synthetic nanoantibody library NanoOri 1.0 (originally from Shanghai Qi Biotechnology Co., Ltd.) was used as a seed library for antibody sequence screening.

[0390] The full-length ROR1 sequence purchased from Sino Biological was constructed and incorporated into an existing lentiviral vector for lentiviral transduction to generate a CHO-ROR1 overexpressing target cell line. Four rounds of panning were performed using the CHO-ROR1 target cell line. A single aliquot of the synthetic nanobody library, NanoOri 1.0, was added with PEG / NaCl and reprecipitated. CHO-ROR1 cells were selected as target cells for panning, while CHO cells were used as negative cells. 500 μL of phage was mixed with 500 μL of 10% FBS / PBS buffer to a final volume of 1 mL in a 2 mL low-binding EP tube. Blocking was performed at 4°C for 1 hour with gentle rotation or periodic mixing. CHO-ROR1 cells were harvested by centrifugation at 500 g for 10 minutes and washed once with 10 mL of PBS. Cells were counted, and when viable cells reached 95% or higher, they were washed three times with 5% FBS / PBS buffer. Then resuspend in 1 mL of 5% FBS / PBS buffer to make the cell number reach 1×10 7 During this time, place the cells on ice at 4°C. Centrifuge CHO-ROR1 cells at 500g for 2 minutes and resuspend in 1 mL of the blocked phage antibody library. Gently rotate or periodically mix at 4°C or room temperature for 2 hours to allow binding. Remove the supernatant, harvest the cells, and resuspend them in 1 mL of 5% FBS / PBS buffer. Repeat this wash twice. After the final resuspension, transfer the cells to a new 2 mL low-binding EP tube to prevent elution of phage that has nonspecifically adsorbed to the EP tube.

[0391] Resuspend the cells in 1 mL of 100 nM Gly-HCl (pH 2.2), incubate at room temperature for 10 minutes, and centrifuge at 500 g for 2 minutes at 4°C. Transfer the supernatant to a fresh EP tube and add 25 μL of 2M Tris for neutralization. Add 500 μL of 7.5% FBS / PBS buffer to the neutralized cells for further neutralization.

[0392] Centrifuge three aliquots of the adsorbed CHO cells and treat them as for the CHO-ROR1 cells. Resuspend the eluted, neutralized phage in the CHO cells. Gently rotate or periodically mix to allow binding for 30 minutes at 4°C. Centrifuge at 500g for 2 minutes at 4°C and use the supernatant for the next adsorption step three times. The final phage supernatant is then used to infect TG1 cells.

[0393] The second round of cell selection was carried out in the same way, and the number of target cells CHO-ROR1 was 5×10 6 The same operation method was used for the third round of cell selection, and the number of cells was 1×10 6The number of washes is increased to 10 times.

[0394] After three rounds of panning, significant enrichment of the output phage was observed. Pool ELISA analysis using cells was performed, and the results are shown in Figure 1. After three rounds of panning, nonspecific enrichment of CHO cells was observed, but binding to CHO-ROR1 was stronger, likely due to an excess of phage, which readily adhered to the cells. Therefore, protein pool ELISA analysis was performed, using ROR1-His as the positive protein and MSLN-His as the nonspecific protein. The results, shown in Figure 2, show significant and specific enrichment of the ROR1-His protein after three rounds of panning, with no binding to the MSLN-His protein.

[0395] The panned phage antibody clones were identified using a protein-based single phage ELISA: 100 μL / well of 1 μg / mL protein was coated on a 96-well ELISA plate at 4°C overnight. Nonspecific binding sites were then blocked with 5% skim milk powder. After thorough washing, the monoclonal phage supernatant (premixed with skim milk powder) was added to the 96-well plate and incubated for 2 hours. After thorough washing, Anti-M13-HRP was added and the reaction was allowed to proceed for 45 minutes. After thorough washing, TMB was added for color development. After 5-10 minutes at room temperature, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450 nm.

[0396] Phage antibody clones that specifically bind to human ROR1 were obtained by ELISA. Single-chain VHH gene sequences were obtained and numbered after sequencing. The obtained antibodies and their sequences are shown in Table 1.

[0397] Table 1 Schematic diagram of antibody sequences targeting ROR1

[0398] Example 2 Construction of anti-ROR1 VHH_hFc fusion antibody and its eukaryotic expression and purification

[0399] The phage antibody clones and the positive control antibody UC961scFv were redesigned and constructed into an Fc (IgG1)-containing expression vector: primers were designed to amplify the VHH / scFv of the phage antibody clones by PCR. The PCR products were then recombinantly cloned into the pcDNA3.4hFc vector, which had been double-digested with SfiI and NotI restriction endonucleases. After sequencing, the plasmids were transfected into Expi293F cells for transient expression and purified using a Protein A affinity column to obtain the antibodies. SEC purity analysis of antibodies 001-010 is shown in Figure 3.

[0400] Example 3 Detection of binding activity of anti-ROR1 antibodies to cell surface ROR1

[0401] The cell surface target antigen (ROR1) and antibody binding activity were detected by flow cytometry fluorescence sorting (FACS) using iQue Screener flow cytometer (purchased from IntelliCyt) using PBS containing 0.1% BSA as a buffer. Briefly, the concentration of the buffer was 1×10 6 Target cells (i.e., SKOV3-ROR1 cells) at a concentration of 1:1 cells / mL were plated onto a 96-well conical-bottom plate (Corning 3894), with 30 μL added to each well. Detection antibody was prepared in buffer at a concentration of 10 μg / mL and diluted three-fold to create a concentration gradient of eight. 30 μL of the prepared antibody at various concentrations was added to the plated target cells at a concentration of 30 μL / well, mixed thoroughly, and incubated at 4°C for 1 hour. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 minutes, the supernatant discarded, and the cells shaken to loosen. Washed again. Fluorescent secondary antibody (ab98593) was prepared in buffer at a 1:200 ratio, added to the cells at a concentration of 30 μL per well, mixed thoroughly, and incubated at 4°C for 30 minutes. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 minutes, the supernatant discarded, and the cells shaken to loosen. Washed again. 35 μL of buffer was added to each well, mixed thoroughly, and analyzed by flow cytometry.

[0402] The results of the flow cytometry affinity binding assay are shown in Figure 4. All of the anti-ROR1 VHHs described herein were detected to have a certain MFI value, indicating that they all bind to ROR1 on the cell surface. At the same antibody concentration, the MFI values ​​of 001, 002, and 003 were significantly higher than those of the control antibody, indicating that 001, 002, and 003 have higher binding affinity for cell surface ROR1 than the positive control.

[0403] Example 4 Construction of Chimeric Antigen Receptor Lentivirus Expression Vector

[0404] Select universal lentiviral vector (Beijing Sino Biological Science and Technology Co., Ltd., Catalog No.: LVCV-01) was used as the CAR lentiviral core empty plasmid (hereinafter referred to as the CAR lentiviral empty vector). The core sequence and Ori element were constructed into the expression region of the CAR lentiviral empty vector to form the sequence of the new vector pcore vector (in-house constructed by Yuanqi Biotechnology Co., Ltd.). DH5α competent cells were transformed, and single colonies were picked and sequenced for identification. The various CAR components and connection sequence in the CAR lentiviral empty vector and ROR1 core plasmid are shown in Figure 5 (using 001 as an example).

[0405] Example 5 Lentivirus packaging and virus titer determination

[0406] As an example, the lentiviral vector system used to construct the present invention belongs to the fourth generation. The system consists of four plasmids, namely the pRH1 plasmid encoding the Gag-Pol protein, the packaging plasmid pMH2 of the Rev protein, the pVH3 plasmid encoding the envelope protein VSV-G, and the core plasmid, i.e., the various CAR lentiviral plasmids containing the VHH sequence in Example 4 above.

[0407] The packaging process of lentivirus is as follows: take cells with good growth status (generally select cells within 20 generations), about 2.5×10 6 293T cells were mixed in 5 mL of complete 293T cell culture medium (high-glucose DMEM with 10% FBS), plated in a 6 cm dish (ensure cell confluency is 80-90% after 20-24 hours), and cultured overnight at 37°C in a 5% CO2 incubator. Approximately 20-24 hours later, 300 μL of Opti-MEM medium was added to a total of 15 μg of the three plasmids (at a mass ratio of pRH1:pMH2:pVH3:core plasmid = 1:1:2:2). After mixing, 45 μL of FuGENEHD transfection reagent (Promega, E2311) was added. After careful mixing, the mixture was allowed to stand at room temperature for 15-20 minutes. The mixture was then carefully added dropwise to the 6 cm dish, the dish was gently shaken, and cultured overnight at 37°C in a 5% CO2 incubator. About 20-24 hours after transfection, carefully discard the supernatant in the 6 cm culture dish, and then slowly add 5 mL of 293T cell complete culture medium.

[0408] About 48 hours after transfection, collect the upper layer of liquid (containing dead cells and debris) in the 6 cm culture dish, centrifuge at 3000g for 5 minutes, transfer the upper layer of virus liquid with cell debris removed to a new centrifuge tube, aliquot, and store at -80℃ until use.

[0409] The lentiviral titer was determined as follows: Rapidly thaw the virus solution prepared above. 293T cells in good growth condition (generally cells cultured for less than 20 generations) were collected, the supernatant was discarded, the cells were washed with PBS, and digested with 0.25% trypsin (GIBICO) at 37°C for approximately 3 minutes. After complete digestion, a certain volume of 293T complete cell culture medium was added to terminate the reaction. Samples were taken and counted, and the cell density was adjusted to 2.0×10 5 / mL, and add polybrene with a final concentration of 10μg / mL, and then inoculate the cells in a six-well plate at an inoculation volume of 2.5mL per well. Then add the above-mentioned virus solution to the six-well plate respectively. Generally, three loading gradients are made for each virus solution: 2μL, 5μL and 10μL, and 1-2 blank wells without virus solution are required as negative controls for flow cytometry detection. The cell culture plate is placed in a 37°C, 5% CO2 incubator for static culture. After the cells have been statically cultured for 48 hours, the upper layer of waste liquid is discarded, the cells are washed with PBS, and digested with 0.25% trypsin (GIBICO) at 37°C for about 3 minutes. After the cells are completely digested, a certain volume of 293T cell complete culture medium is added to terminate the reaction, samples are taken and counted, and then about 5×10 5 Transfer 100 cells to a sterile 1.5mL centrifuge tube and label it. Fill to 1.2-1.5mL with PBS solution (PBS solution containing 1% newborn calf serum), centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant. Add 1mL of PBS to each tube, gently pipette to mix, centrifuge at 500g for 5 minutes at 4°C, and thoroughly discard the supernatant. Add 50μL of PBS and 0.8μg of biotin-goat anti-human F(ab)2 antibody (109-066-006) to each tube, gently pipette to mix (for negative control, no primary antibody was added), and incubate at 4°C for 60 minutes. After incubation, directly resuspend the cells in 1mL of PBS, centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant. Add 50 μL of PBS and 0.5 μg of APC streptavidin (554067) fluorescent secondary antibody to each tube (omit this protein for negative control) and incubate at 4°C for 30 min. Resuspend cells in 200 μL of PBS per tube. Fluorescence expression percentages were determined on a BD FACS Canto II flow cytometer and data processed. Results are valid only when the positive rate in each test tube minus the positive rate of the negative control is 5% to 20%.

[0410] Lentivirus titer (TU / mL) = number of plated cells × (positive rate of test tube - positive rate of control tube) / volume of inoculated virus solution (mL).

[0411] After testing, the titer of each VHH-containing CAR virus ranged from 1 to 10×10 6 TU / mL.

[0412] Example 6 Infection of T cells and in vitro expansion of ROR1 CAR-T cells

[0413] The preparation method of CAR-T cells containing ROR1 antibody sequence and positive control sequence UC961 scFv is as follows: Human peripheral blood mononuclear cells (PBMCs) are isolated from blood samples collected by apheresis by Ficoll density gradient centrifugation. PBMCs are sorted with CD3 positive magnetic beads to obtain CD3+ T cells with a purity of >90%. The specific method of T cell sorting is shown in the product manual (MACS, DS130-050-101). CD3+ T cells are resuspended in T cell complete medium (X-VIVO 15 (Lonza) + 5% FBS + cytokines), and washed CD3 / CD28 Dynabeads magnetic beads (Gibco, 40203D) are added at 3 times the cell volume. Then, T cell complete medium is added to adjust the cell density to 1.0-1.2×10 6 cells / mL, and the cells were placed in a 37°C, 5% CO2 incubator for activation culture (recorded as D0). Generally, lentiviral transduction was performed 20-24 hours after T cell activation. T cells activated for 20-24 hours were collected, centrifuged at 500g for 5 minutes, and resuspended in a certain volume of T cell complete medium. After counting, the virus solution with the titer determined in Example 5 was added according to the virus counterstaining index (MOI) of 2-5. Then, polybrene was added to a final concentration of 5 μg / mL, and T cell complete medium was added to adjust the cell density to 0.6-1.0×10 6 / mL, and then culture the cells in a 37°C, 5% CO2 incubator. Another aliquot of T cells was not infected with lentivirus and served as a negative control group. About 20-24 hours after viral transduction, T cells from each group were collected and centrifuged at 500g for 5 minutes. After resuspending the cells in a certain volume of T cell complete medium, a sample was taken for counting and replenishing T cell complete medium to adjust the cell density to 0.5-0.7×10 6 cells / mL. After that, the CAR-T cells were counted every 1-2 days, and T cell complete culture medium was added according to the actual situation to adjust the cell density to 0.5-1.0×10 6 cells / mL. Generally, Dynabeads magnetic beads are removed on the 7th day of culture, and the total culture period is about 12 days.

[0414] The cumulative expansion folds are shown in Figure 6. After 13 days of activation and culture, the total expansion folds of ROR1 CAR-T cells ranged from 100 to 400. The positive rate results are shown in Figure 7, indicating that the prepared CAR-T cells can be used in cytological function experiments.

[0415] Example 7 Evaluation of in vitro targeted killing activity of ROR1 CAR-T cells

[0416] To accurately assess the cytotoxicity of CAR-T cells against target cells, we selected SKOV3 and CaoV3 cells, which naturally express ROR1, as target cells. Effector cells cultured on day 9 were incubated with target cells for 20 hours at effector-target ratios of 1:1, 1:3, and 1:9. Target cell survival (i.e., CAR-T cell cytotoxicity) was assessed using the LDH assay.

[0417] The cell cytotoxicity can be calculated using the following formula:

[0418] Killing rate (%) = (experimental well - spontaneous release of target cells - spontaneous release of effector cells) / (maximum release of target cells - spontaneous release of target cells) × 100%.

[0419] The results, as shown in Figures 8A and 8B, show that compared to blank effector T cells, both anti-ROR1 CAR-T cells and the UC961 positive control showed significant cytotoxicity against target cells, and this was gradient-dependent, indicating that the CAR-T cells prepared with the antibodies of the present application have specific binding and significant cytotoxicity against target cells expressing ROR1. For SKOV3 target cells, CAR-T cells prepared with 001 and 002 showed stronger cytotoxicity than the positive control. For CaoV3 target cells, CAR-T cells prepared with 001, 002, and 004 showed stronger cytotoxicity than the positive control.

[0420] Example 8 Cytokine Secretion after ROR1 CAR-T Cell Killing in Vitro

[0421] Referring to Example 7, a target cell killing experiment was performed in a 96-well plate, with the ratio of effector cells to target cells being 1:1, and then 2×10 effector cells were fixed in each 96-well plate. 4 Target cells and effector cells were added in sequence, and two replicate wells were set up for each effector-target ratio in each group. In addition, two separate effector cell wells were set up in each group to detect the background factor secretion of the effector cells.

[0422] About 24 hours after the target cells were killed, the 96-well plate was centrifuged and the supernatant of each well was collected (if not tested immediately, the supernatant should be stored in a -80°C refrigerator).

[0423] The effector cells were centrifuged and the supernatant was removed. The cells were diluted to 3×10 5 cells / mL, and 100 μL was used per well. CAR-T cells without target cells (i.e., CAR-T cells not co-cultured with tumor cells) and target cells were mixed in a round-bottom 96-well plate at a 1:1 effector-target ratio. After incubation for 24 hours, the supernatant was aspirated and the levels of IL-2 and IFN-γ in the supernatant were measured using the R&D Cytokine Kit.

[0424] The experimental results are shown in Figures 9A-9D. Without target cell stimulation, each group of CAR-T cells secreted a small amount of IL-2 and IFN-γ. When stimulated with target cells SKOV3 and CaoV3, the positive control UC961 selected by this application secreted almost no change in IL-2 and IFN-γ when killing target cells. In contrast, when stimulated with target cells SKOV3, the 001, 002, 004, and 005 CAR-T cells of this application produced more IFN-γ when killing target cells; when stimulated with target cells CaoV3, the 001, 002, and 004 CAR-T cells of this application produced more IFN-γ when killing target cells; and when stimulated with target cells SKOV3 and CaoV3, the 001 and 002 CAR-T cells of this application produced more IL-2 when killing target cells. Experimental results show that CAR-T cells prepared with some antibodies in this application can secrete more cytokines under the stimulation of target cells.

[0425] Example 9 Targeted Proliferation Ability of ROR1 CAR-T Cells

[0426] In this example, the targeted proliferation capacity of CAR-T cells was tested using natural SKOV3 cells as target cells. After approximately 9-12 days of CAR-T cell culture, the CAR-T cells and SKOV3 cells were incubated at a 1:1 effector-target ratio for the first round of targeted stimulation. After 4-5 days of co-incubation, all cells were collected, sampled, and counted. A second round of targeted stimulation was then performed at a 1:3 (CAR-T cell:new SKOV3 cell) ratio. After another 4-5 days of co-incubation, a third round of targeted stimulation was performed (at the same ratio as in the second round). During these three rounds of targeted stimulation, appropriate amounts of X-VIVO 15 medium were added every 1-2 days based on cell growth.

[0427] The experimental results are shown in Figure 10. After two or three rounds of targeted stimulation of ROR1 CAR-T cells, the CAR-T cells prepared by the antibodies of this application all expanded. The number of cells after stimulation exceeded that before stimulation, and the proliferation of 001 and 002 CAR-T cells exceeded that of the positive control. The T cell co-incubation wells and the single SKOV3 target cell wells basically did not expand. The experimental results show that all CAR-T cells prepared by the antibodies of this application have targeted proliferation capabilities.

[0428] Example 10 In vivo experiments in ROR1 CAR-T mice

[0429] We further verified the tumor-suppressing effect of ROR1 CAR-T cells in NSG mice. Specifically, the experiment was divided into five groups: T cell group, positive control group, and ROR1 CAR-T cell group (001 and 002), with 5 mice in each group. First, 3×10 6 SKOV3 cells were inoculated at a dose of 10 cells / mouse. After about 2 weeks (tumor size was 80 mm 3 ) and intratumoral injection of frozen and revived CAR-T (or Mock T) cells, with a dose of 5×10 6 The dose of CAR-T cells per mouse was adjusted to the total number of cells inoculated per mouse using T cells. After inoculation of CAR-T (or T) cells, the tumor size and body weight of the mice were measured three times a week and observed for three weeks.

[0430] The experimental results of tumor inhibition speed are shown in Figure 11A. The tumor inhibition effect of 001 and 002 CAR-T cells is better than that of the positive control group. At the same number of days after injection, the tumor volume is smaller, indicating that 001 and 002 CAR-T cells have a significant tumor inhibition effect.

[0431] The in vivo safety results are shown in Figure 11B. After B-NDG mice were inoculated with CAR-T (or T) cells, they were weighed every 2-3 days, and a weight curve was plotted. Coat color, excrement, food and water intake, body movement, and mortality were observed and recorded. The weight of mice in the experimental group remained stable, consistent with the weight curve trend of mice in the mock T group. During the experiment, mice in the ROR1 CAR-T cell and positive control groups had shiny fur, and their behavior, food and water intake, and urine and stool color were normal, confirming the good safety of 001 and 002 CAR-T cells in mice.

[0432] Example 11 ROR1 Antibody Endocytosis Effect Experiment

[0433] SKOV3-ROR1 cells overexpressing ROR1 target antigen were selected as follows: 5×10 4Target cells were plated into a 96-well V-bottom plate at 30 μL / well. The antibody dilution concentration started at 20 μg / mL and was diluted 3-fold in a series of 7 concentration gradients. 30 μL / well was added to the 96-well V-bottom plate with target cells, mixed and incubated for 1 hour, and washed once with PBS containing 0.1% BSA. Referring to the antibody dilution concentration, anti-human IgG-Fc (Dylight650) secondary antibody was added at a dilution of 1:200 at 30 μL / well. After incubation at 4°C for 30 min, the plates were washed once with PBS containing 0.1% BSA. 50 μL of complete culture medium (RPMI1640 + 10% FBS) was added, and plates A and B were incubated in a 37°C incubator for 2 hours, respectively, and washed once with PBS containing 0.1% BSA. 30 μL of fluorescence quenching buffer was added to each well, and the plates were placed at 4°C for 10 min. The plates were then washed once with PBS containing 0.1% BSA, and resuspended in PBS containing 0.1% BSA at 30 μL / well for detection by Intellicyt iQue3 flow cytometer.

[0434] The experimental results are shown in Figures 12A and 12B. The ROR1 antibodies provided in this application all have different degrees of endocytosis effect at 37°C. Compared with the control, the endocytosis effect of 001 and 007 antibodies is better than that of the positive control antibody, and 001 antibody has the best endocytosis effect.

[0435] Example 12 ROR1 CAR-T cell killing in vitro

[0436] To test the cytotoxicity of all CAR-T cell sequences against target cells, we selected SKOV3 and MDA-MB-231 cells, which naturally express ROR1, as target cells. Effector cells cultured to day 9 were incubated with target cells at a 1:1 ratio for 20 hours. Target cell survival (i.e., CAR-T cell cytotoxicity) was assessed using the LDH assay.

[0437] The cell cytotoxicity can be calculated using the following formula:

[0438] Killing rate (%) = (experimental well - spontaneous release of target cells - spontaneous release of effector cells) / (maximum release of target cells - spontaneous release of target cells) × 100%.

[0439] The experimental results are shown in Figures 13A, 13B, and 13C. Compared with blank effector T cells, the anti-ROR1 CAR-T cells of the present application have a higher killing effect on SKOV3 or MDA-MB-231 target cells than blank effector T cells. The results show that the anti-ROR1 CAR-T cells of the present application have a significant killing effect on target cells expressing ROR1.

Claims

1. An isolated antigen-binding protein capable of binding to ROR1, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NOs:65-70, SEQ ID NOs:72-77, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NOs:85-92 and SEQ ID NOs:129-132.

2. The antigen-binding protein according to claim 1, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 62-92.

3. The antigen binding protein according to any one of claims 1-2, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 33, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NOs: 144-155.

4. The antigen-binding protein according to any one of claims 1 to 3, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 29 to 61.

5. The antigen binding protein according to any one of claims 1 to 4, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 24 and SEQ ID NOs: 133-143.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 28.

7. The antigen binding protein according to any one of claims 1 to 6, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from any one of the following combinations: (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 62; (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63; (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 64; (4) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 66; (6) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67; (7) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67; (8) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 71; (9) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 40, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 67; (10) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 72; (11) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 73; (12) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 36, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 68; (13) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 43, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 74; (14) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 64; (15) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 45, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 75; (16) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 69; (17) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 76; (18) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 46, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 77; (19) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 78; (20) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 70; (21) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79; (22) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 80; (23) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 81; (24) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67; (25) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 52, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 82; (26) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83; (27) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 53, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 84; (28) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 83; (29) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85; (30) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 86; (31) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 56, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 87; (32) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 57, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 88; (33) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 89; (34) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 90; (35) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 91; and (36) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:61, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

92.

8. The antigen binding protein according to any one of claims 1 to 7, wherein the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4.

9. The antigen-binding protein according to any one of claims 1 to 8, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 93 to 128.

10. The antigen binding protein according to any one of claims 1 to 9, wherein the antigen binding protein further comprises an immunoglobulin constant region. The antigen binding protein according to claim 10 , wherein the immunoglobulin constant region is an immunoglobulin Fc region.

12. The antigen binding protein according to any one of claims 10-11, wherein the immunoglobulin constant region is an IgG Fc region.

13. The antigen binding protein according to any one of claims 10 to 12, wherein the immunoglobulin constant region is a human IgG Fc region.

14. The antigen binding protein according to any one of claims 1 to 13, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

15. The antigen-binding protein of claim 14, wherein the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

16. The antigen binding protein according to any one of claims 1 to 15, wherein the antigen binding protein is a chimeric antibody, a humanized antibody or a fully human antibody.

17. The antigen binding protein according to any one of claims 1 to 16, wherein the antigen binding protein is a monospecific antibody, a bispecific antibody or a multispecific antibody.

18. The antigen binding protein according to any one of claims 1 to 17, wherein the antigen binding protein is a monovalent antibody, a bivalent antibody or a multivalent antibody.

19. A chimeric antigen receptor comprising the antigen binding protein of any one of claims 1 to 18.

20. The chimeric antigen receptor of claim 19, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

21. The chimeric antigen receptor of claim 20, wherein the antigen binding domain comprises the antigen binding protein of any one of claims 1-18.

22. The chimeric antigen receptor of any one of claims 20-21, wherein the transmembrane domain comprises a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, 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, CD137, CD154, and SLAM.

23. The chimeric antigen receptor of any one of claims 20-22, wherein the transmembrane domain is derived from the transmembrane domain of CD8.

24. The chimeric antigen receptor of any one of claims 20-23, wherein the costimulatory domain comprises a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, 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.

25. The chimeric antigen receptor of any one of claims 20-24, wherein the costimulatory domain is derived from a 4-1BB costimulatory domain.

26. The chimeric antigen receptor of any one of claims 20-25, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from any one of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

27. The chimeric antigen receptor of any one of claims 20-26, wherein the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ.

28. The chimeric antigen receptor of any one of claims 20-27, wherein the chimeric antigen receptor further comprises a hinge region.

29. The chimeric antigen receptor of claim 28, wherein the hinge region comprises a hinge region derived from any one of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

30. The chimeric antigen receptor of any one of claims 20-29, wherein the hinge region is derived from the hinge region of CD8.

31. A modified immune cell, wherein the immune cell comprises the chimeric antigen receptor of any one of claims 19-30.

32. The modified immune cell of claim 31, wherein the immune cell is a T cell, a NK cell, an iNKT cell, a dendritic cell and / or a macrophage.

33. The modified immune cell according to any one of claims 31-32, further comprising and / or expressing a low-density lipoprotein receptor-related protein or a fragment thereof.

34. The modified immune cell according to claim 33, 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.

35. The modified immune cell according to any one of claims 33-34, wherein the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

36. The modified immune cell according to any one of claims 33-35, wherein the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

156.

37. The modified immune cell according to any one of claims 33-36, wherein the low-density lipoprotein receptor-related protein or a fragment thereof is an exogenous low-density lipoprotein receptor-related protein or a fragment thereof.

38. An immunoconjugate, wherein the immunoconjugate comprises the antigen binding protein of any one of claims 1-18.

39. The immunoconjugate of claim 38, wherein the immunoconjugate comprises an antigen binding protein, a linker, and a payload.

40. The immunoconjugate of claim 39, wherein the payload is a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid.

41. The immunoconjugate of any one of claims 38-40, wherein the immunoconjugate is an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted nuclear drug (RDC).

42. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1-18 and / or the chimeric antigen receptor of any one of claims 19-30.

43. A vector comprising the nucleic acid molecule of claim 42.

44. A cell comprising the nucleic acid molecule of claim 42 and / or the vector of claim 43.

45. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-30, the modified immune cell of any one of claims 31-37, the immunoconjugate of any one of claims 38-41, the nucleic acid molecule of claim 42, the vector of claim 43 and / or the cell of claim 44, and optionally a pharmaceutically acceptable carrier.

46. A detection kit comprising the antigen binding protein according to any one of claims 1-18, wherein the detection kit is used to detect the presence and / or content of ROR1 in a sample or a subject.

47. Use of the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-30, the modified immune cell of any one of claims 31-37, the immunoconjugate of any one of claims 38-41, the nucleic acid molecule of claim 42, the vector of claim 43, the cell of claim 44 and / or the pharmaceutical composition of claim 45 in the preparation of a detection kit for diagnosis.

48. A method for detecting the presence and / or content of ROR1, comprising using the antigen binding protein of any one of claims 1-18.

49. Use of the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-30, the modified immune cell of any one of claims 31-37, the immunoconjugate of any one of claims 38-41, the nucleic acid molecule of claim 42, the vector of claim 43, the cell of claim 44 and / or the pharmaceutical composition of claim 45 in the preparation of a medicament for preventing, diagnosing and / or treating a disease and / or condition.

50. The use according to claim 49, wherein the disease and / or disorder is a tumor.

51. The use according to any one of claims 49-50, wherein the tumor is a solid tumor and / or a hematological tumor.

52. The use according to any one of claims 49-51, wherein the tumor is a ROR1 positive tumor.

53. The use according to any one of claims 49 to 52, wherein the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer or colorectal cancer.

54. A method for preventing, diagnosing and / or treating a disease and / or condition, comprising administering to a subject in need thereof the antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-30, the modified immune cell of any one of claims 31-37, the immunoconjugate of any one of claims 38-41, the nucleic acid molecule of claim 42, the vector of claim 43, the cell of claim 44 and / or the pharmaceutical composition of claim 45.

55. The method of claim 54, wherein the disease and / or condition is a tumor.

56. The method of any one of claims 54-55, wherein the tumor is a solid tumor and / or a hematological tumor.

57. The method of any one of claims 54-56, wherein the tumor is a ROR1 -positive tumor.

58. The method of any one of claims 54-57, wherein the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer, or colorectal cancer.

59. The antigen binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-30, the modified immune cell of any one of claims 31-37, the immunoconjugate of any one of claims 38-41, the nucleic acid molecule of claim 42, the vector of claim 43, the cell of claim 44 and / or the pharmaceutical composition of claim 45, for use in preventing, diagnosing and / or treating a disease and / or disorder.

60. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition of claim 59, wherein the disease and / or disorder is a tumor.

61. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition of any one of claims 59-60, wherein the tumor is a solid tumor and / or a hematological tumor.

62. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition of any one of claims 59-61, wherein the tumor is a ROR1-positive tumor.

63. The antigen binding protein, chimeric antigen receptor, modified immune cell, immunoconjugate, nucleic acid molecule, vector, cell and / or pharmaceutical composition of any one of claims 59-62, wherein the tumor is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, mesothelioma, endometrial cancer, pancreatic cancer, gastric cancer, esophageal cancer, sarcoma, kidney cancer, liver cancer, thyroid cancer or colorectal cancer.

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