Single-domain antibodies targeting human ROR1

Abstract

The present specification provides a single domain antibody that targets the ROR1 protein, and a chimeric antigen receptor (CAR) and CAR-T cells comprising the single domain antibody. The present specification further provides applications of the single domain antibody and CAR-T cells in the treatment of tumors that express the ROR1 protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Chinese patent application having application number 202211370878.5 and filing date November 3, 2022, and Chinese patent application having application number 202310575171.6 and filing date May 18, 2023, which are incorporated herein by reference in their entireties. [Technical Field]

[0002] The present invention relates to the fields of tumor immunotherapy and molecular immunology, and specifically to the development of human ROR1-targeting CAR-T with high affinity, high specificity, multiple antigen-recognizing epitopes, and higher functionality. [Background technology]

[0003] Tumor immunotherapy has become one of the most important means of tumor treatment. The immune system can only effectively attack cancer cells once it has identified them. Malignant transformation is the result of normal somatic cells losing their normal cell control function and accumulating a certain amount of genetic mutations. However, cancer cells can cleverly evade the immune system's attack by disguising themselves as normal somatic cells.

[0004] Tumor-associated antigens (TAAs) refer to self-antigens expressed by tumor cells. They are partially present in normal host cells, primarily resulting from gene amplification or post-translational modification, but tend to be highly or specifically expressed in tumor cells. Currently, many tumor-associated and tumor-specific antigens have been discovered, and targeted therapy targeting TAA has become an important approach to cancer treatment. Several cancer immunotherapeutic drugs based on this mechanism are already on the market and have proven highly effective in clinical trials.

[0005] Monoclonal antibodies (mAbs) have become an increasingly important drug class, and their clinical applications have completely transformed the field of cancer treatment. Different mAbs have different mechanisms of antitumor action, including blockade of tumor-specific growth factor receptors or immunoregulatory molecules, and complement- and cell-mediated tumor cell lysis. Rituximab targets the CD20 antigen on B cells to treat non-Hodgkin's lymphoma. Trastuzumab (Herceptin) targets HER2 to treat HER2-positive breast cancer—these are just two well-known examples. Drug development against TAAs remains a very active area of ​​research. Currently, a significant number of targeted drugs for different tumor types are in preclinical or clinical trials.

[0006] Generally, antibody-mediated TAA recognition can effectively induce NK cells or T cells to target tumor cells with high expression of the corresponding TAA. In contrast, antibodies against TAA (e.g., rituximab and trastuzumab) can not only directly kill tumor cells through ADCC effectors, but also be used as diagnostic markers or to innovatively increase the targeting of conventional cancer therapies.

[0007] Adoptive immunotherapy is an innovative treatment in hematology that involves genetically engineering T cells to express synthetic chimeric antigen receptors (CARs). CARs are engineered receptors that can redirect immune cells to target cancer cells. CAR-T cells have demonstrated impressive efficacy in patients with hematological malignancies, and the FDA has approved six CAR-T cells for treating relapsed or refractory B-cell malignancies: Abecma, Breyanzi, Carvykti, Kymriah, Tecartus, and Yescarta. The components of these CARs (extracellular antigen-binding domain, hinge and transmembrane region, costimulatory domain, and activation domain) are systematically engineered to optimize cell activation and enhance cell persistence. The use of different domains or subtle modifications to the domain sequence can significantly alter the efficacy of CAR-T cells. While CAR-T has been successful in treating hematologic malignancies, many patients experience CAR-T cell-associated toxicity and relapse after CAR-T cell therapy. This may be related to the CAR's binding epitope, suggesting the need to develop a variety of antibodies that bind to the epitope. Next-generation CAR-T cells aim to reduce toxicity and prevent relapse by refining antigen targeting, modulating the assembly or activation of CAR components, preventing anti-CAR immunity, and / or protecting cells to respond to their surrounding environment. While FDA-approved CAR-T cells target two B cell antigens, CD19 and B-cell maturation antigen (BCMA), others targeting B cell malignancies, other hematologic cancers, and solid tumors are rapidly emerging. The development of new CAR-T therapies is urgently needed to address unmet clinical needs, particularly in the case of solid tumors.

[0008] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein and an orphan receptor tyrosine kinase-like surface antigen expressed in many tissues during embryonic development and functions in embryonic skeletal, cardiopulmonary, and neural development. ROR1 is primarily expressed in embryonic tissues, with limited expression levels in adult tissues, including the parathyroid gland, pancreas, esophagus, stomach, and duodenum. ROR1 is expressed in many B-cell malignancies and various cancer cell lines, including solid tumors such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and ovarian cancer. ROR1 is expressed on the surface of CLL cells and is an accurate and reliable marker of CLL minimal residual disease. High ROR1 expression levels are associated with high circulating leukemia cells and disease aggressiveness, and ROR1 expression is maintained in CLL cells during and after treatment. Therefore, ROR1 is an ideal drug target for cancer therapy (Balakrishnan et al., Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues, Clin Cancer Res (2017) 23 (12): 3061-3071; Aghebati-Maleki et al., Receptor tyrosine kinase-like orphan receptor 1 (ROR-1): An emerging target for diagnosis and therapy of chronic lymphocytic leukemia, Biomedicine & Pharmacotherapy, Volume 88, April 2017, Pages 814-822; De Propris et al., ROR1 is an accurate and reliable marker of minimal residual disease in chronic lymphocytic leukemia, British Journal of Haematology, Volume 190, Issue 6, September 2020, Pages e346-e349). Summary of the Invention

[0009] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that targets a ROR1 protein, wherein the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from one of the following combinations: (1) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:2; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:4; (2) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:7; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:8; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:9; (3) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 12; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 13; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 14; (4) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 17; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 18; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (5) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 22; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:23; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:24; (6) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 27; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:28; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:29; (7) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 32; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:33; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:34, and (8) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 37; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:38; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:39; (9) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:67; (10) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:71; (11) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:4; (12) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 75; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (13) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 78; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (14) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 80; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (15) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 82; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:83; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (16) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 85; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (17) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 85; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:87; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; (18) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 89; the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:90; the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; Or, The antibody is a mutant of an antibody defined by the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of (1) to (18), wherein the mutant contains a total of 1 to 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid modifications in the HCDR1, HCDR2, and HCDR3 sequences compared to the antibody defined in any one of (1) to (18).

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is: (1) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 1 or at least 90% sequence identity thereto; (2) a heavy chain variable region sequence set forth in SEQ ID NO: 6 or having at least 90% sequence identity thereto; (3) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 11 or at least 90% sequence identity thereto; (4) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 16 or at least 90% sequence identity thereto; (5) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 21 or at least 90% sequence identity thereto; (6) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 26 or at least 90% sequence identity thereto; (7) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 31 or at least 90% sequence identity thereto; (8) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 36 or at least 90% sequence identity thereto; (9) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 65 or at least 90% sequence identity thereto; (10) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 68 or at least 90% sequence identity thereto; (11) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 70 or at least 90% sequence identity thereto; (12) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 81 or at least 90% sequence identity thereto; (13) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 84 or at least 90% sequence identity thereto; (14) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 86 or at least 90% sequence identity thereto; and (15) A heavy chain variable region sequence selected from the sequence shown in SEQ ID NO: 88 or a heavy chain variable region sequence having at least 90% sequence identity thereto.

[0011] In some embodiments, the antibody is a single domain antibody.

[0012] In some embodiments, the antibody is a humanized antibody.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of the humanized antibody is: (1) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 41 or at least 90% sequence identity thereto; (2) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 43 or at least 90% sequence identity thereto; (3) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 45 or at least 90% sequence identity thereto; (4) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 72 or at least 90% sequence identity thereto; (5) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 73 or at least 90% sequence identity thereto; (6) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 74 or at least 90% sequence identity thereto; (7) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 77 or at least 90% sequence identity thereto; and (8) A heavy chain variable region sequence selected from the sequence shown in SEQ ID NO: 79 or a heavy chain variable region sequence having at least 90% sequence identity thereto.

[0014] In some embodiments, the antibody targets the Frizzled domain or the Kringle domain of the ROR1 protein.

[0015] In some embodiments, the EC20 value of the binding of the antibody to a ROR1 protein, a Frizzled domain thereof, or a Kringle domain thereof as measured by ELISA. 50 The value is below 0.1 μg / mL.

[0016] In some embodiments, the KD value of the binding of the antibody to ROR1 protein, its Frizzled domain, or its Kringle domain, as measured by surface plasmon resonance technology, is 10 -6 M or less, preferably 10 -7 M or less, and more preferably 10 -8 It is below M.

[0017] In some embodiments, the antibody further comprises an Fc fragment, preferably the Fc fragment is derived from human IgG, eg, IgG1.

[0018] In another aspect, the present disclosure provides a fusion protein comprising at least one antigen-binding functional moiety, wherein the antigen-binding functional moiety comprises an antibody or antigen-binding fragment thereof as described above.

[0019] In some embodiments, the fusion protein comprises at least two antigen-binding functional moieties, each targeting the same or different antigen epitopes.

[0020] In some embodiments, one of the two antigen-binding functional moieties targets the Frizzled domain of the ROR1 protein and another of the two antigen-binding functional moieties targets another part of the ROR1 protein other than the Frizzled domain; one of the two antigen-binding functional moieties targets the Kringle domain of ROR1 and another of the two antigen-binding functional moieties targets another part of the ROR1 protein other than the Kringle domain; or one of the two antigen-binding functional moieties targets the Kringle domain of ROR1 and another of the two antigen-binding functional moieties targets the Frizzled domain of ROR1.

[0021] In some embodiments, the antigen-binding functional moieties are linked together via a peptide linker molecule.

[0022] In another aspect, the description provides a chimeric antigen receptor that targets the ROR1 protein, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof, or the fusion protein described above.

[0023] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 56-63.

[0024] In another aspect, the present specification provides a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof, fusion protein, or chimeric antigen receptor.

[0025] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44, and 46.

[0026] In another aspect, the present specification provides an expression vector, which comprises the above-described nucleic acid molecule.

[0027] In another aspect, the present specification provides a host cell, which comprises the above-mentioned expression vector or expresses the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, or chimeric antigen receptor.

[0028] In some embodiments, the host cell is an immune effector cell and expresses the chimeric antigen receptor.

[0029] In some embodiments, the immune effector cells are T cells or NK cells.

[0030] In another aspect, the present specification provides a pharmaceutical composition comprising: 1) the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, or host cell; and 2) a pharmaceutically acceptable carrier.

[0031] In another aspect, the present specification provides a method of treating a disease, comprising administering to a subject in need thereof an effective amount of the above-described antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, host cell, or pharmaceutical composition.

[0032] In some embodiments, the disease is a tumor that expresses the ROR1 protein.

[0033] In some embodiments, the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma, and ovarian cancer.

[0034] In another aspect, the present specification provides a kit for detecting ROR1 protein in a sample, the kit comprising the above-described antibody or antigen-binding fragment thereof, or the above-described fusion protein.

[0035] In another aspect, the present specification provides the use of the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule or host cell in the manufacture of a medicament for treating a tumor.

[0036] In some embodiments, the disease is a tumor that expresses the ROR1 protein.

[0037] In some embodiments, the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma, and ovarian cancer. [Brief explanation of the drawings]

[0038] [Figure 1] This shows the results of an ELISA experiment between purified monoclonal antibodies and human ROR1-His recombinant protein. [Figure 2] This shows the results of an ELISA experiment between purified monoclonal antibodies and human ROR1 Kringle-His recombinant protein. [Figure 3] This shows the results of an ELISA experiment between purified monoclonal antibodies and human ROR1-His recombinant protein. [Figure 4] 1 shows the results of a FACS experiment of binding between purified monoclonal antibodies and cell lines expressing human and mouse ROR1. [Figure 5] These are the results of affinity experiments between humanized antibodies and their corresponding ROR1-related proteins. [Figure 6] 1 shows the results of a FACS experiment of the binding of humanized antibodies to cell lines expressing human and mouse ROR1. [Figure 7] FIG. 1 shows the results of flow cytometry of the human ROR1 modified cell line CHO-K1 / ROR1. [Figure 8] FIG. 1 is a structural schematic diagram of a constructed CAR of the present invention. [Figure 9] FIG. 1 shows the percentage of T cells by flow cytometry after 4 days of activation of PBMCs. [Figure 10] This is a reporter gene-based evaluation of the activating effect of human ROR1-CAR targeting Jurkat cells. [Figure 11] This is an evaluation of the activating effect of human ROR1-CAR on Jurkat cells as targets, based on the secretion level of the cytokine IL-2. [Figure 12] This figure shows the specific killing effect of human ROR1-CAR-T on the target cell CHO-K1 / ROR1 / Luc. [Figure 13] Figure showing detection of cytokine release levels after co-incubation of human ROR1-CAR-T targets with target cells CHO-K1 / ROR1 / Luc. [Figure 14] This shows the results of an affinity experiment between the AHP15485-VHH4 series affinity matured antibodies and their corresponding ROR1-related proteins. [Figure 15] This shows the results of an affinity experiment between the AHP15662-VHH4 series affinity matured antibodies and their corresponding human ROR1-related proteins. DETAILED DESCRIPTION OF THE INVENTION

[0039] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0040] The term "or" refers to each individual element of a list of alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed alternative elements.

[0041] The term "about" typically refers to a variation within 0.5% to 10% above or below the specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0042] The term "comprises" or "comprises" means including the stated element, integer, or step, but not excluding any other element, integer, or step. As used herein, when the term "comprises" or "comprises," it also covers cases consisting of the stated element, integer, or step, unless otherwise specified. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is intended to cover an antibody variable region consisting of that specific sequence.

[0043] ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved membrane protein widely expressed during embryonic development and in various human cancers. Its molecular structure includes an extracellular domain consisting of an Ig-like domain, a Frizzled domain, and a juxtamembrane Kringle domain; a transmembrane domain; a cytoplasmic domain consisting of a tyrosine kinase-like domain, two serine / threonine-rich domains, and a proline-rich domain (PRD). Due to its expression pattern and function in tumor progression, ROR1 has become a promising target for tumor therapy. ROR1 proteins include human and mouse ROR1 proteins.

[0044] As used herein, the term "antibody" is used in its broadest sense and refers to a protein or polypeptide that exhibits binding specificity to a specific antigen, including immunoglobulins or other corresponding molecules that contain one or more antigen-binding domains that specifically bind to that antigen. Specific examples of antibodies may include intact antibodies (e.g., classical four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, etc. Classical antibody molecules are typically tetramers consisting of two identical heavy chains and two identical light chains linked to each other via disulfide bonds. Conservative differences in amino acid sequence separate the heavy and light chains into an amino-terminal variable region (V) and a carboxy-terminal constant region (C). The variable region is responsible for antigen recognition and binding, while the constant region (e.g., Fc fragment) is responsible for initiating downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy and light chain variable regions each contain three local regions of amino acid composition with a higher degree of sequence variability, which are important sites for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequences of CDRs can be easily determined using numbering schemes well known in the art, such as Kabat, Chothia, IMGT, AbM, or Contact. The three complementarity-determining regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, respectively, and the three complementarity-determining regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, respectively. Each heavy chain variable region (VH) and light chain variable region (VL) may be composed of three CDRs and four FR regions, which may be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In one specific embodiment, the CDR sequences of the antibodies described herein have been determined based on the Kabat numbering scheme.

[0045] An "antigen-binding fragment" of an antibody molecule refers to a polypeptide that contains a partial sequence (especially the CDR sequence) of the original antibody and has the original antibody-binding specificity. Such an antigen-binding fragment usually contains at least the heavy chain variable region of the original antibody and retains antigen-binding ability. Antigen-binding fragments come in various formats, such as Fab, Fab', F(ab')2, single-chain antibodies (scFv), and single-domain antibodies (sdAb). Those skilled in the art already know how to obtain these antigen-binding fragments. For example, a classic antibody molecule can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2, and then treated with a reducing agent to cleave the disulfide bond between the F(ab')2 hinge region to form Fab' fragments. A "single-chain antibody (scFv)" is constructed by linking the antibody heavy chain variable region and light chain variable region via a short peptide to form a peptide chain. With correct folding, the variable regions from the heavy and light chains interact non-covalently to form the Fv segment, allowing scFvs to better retain their avidity against their antigens.

[0046] A "single domain antibody (sdAb)" or "V H Also known as "H antibodies," single domain antibodies refer to antibody molecules that have antigen-binding ability and contain a heavy chain variable region but no light chain. From a structural perspective, single domain antibodies may be fragments of a classical four-chain antibody molecule. Single domain antibodies were first discovered in camelids, and researchers subsequently discovered many more single domain antibodies with antigen-binding ability through screening of antibody libraries (e.g., phage display libraries). Single domain antibodies have several advantages over conventional antibody molecules (e.g., classical antibody molecules), including, but not limited to, their smaller molecular weight, which allows them to reach tissues or sites in the human body that are difficult for conventional antibody molecules to reach, or to access antigen epitopes in proteins or polypeptides that are difficult for conventional antibody molecules to access, and their greater stability, including, but not limited to, their resistance to temperature and pH changes and the action of denaturants and proteases.

[0047] The term "fusion protein" refers to a protein molecule composed of at least two different peptide segments that are artificially generated (e.g., by genetic engineering techniques). These peptide segments do not occur in nature or in the same protein molecule. Examples of common fusion proteins containing antibody fragments include multispecific antibodies, antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also called immunotoxins), enzyme-labeled antibodies for immunodetection, chimeric antigen receptors (CARs), etc. In one specific example, the fusion protein is a multispecific antibody, which comprises at least two single domain antibodies according to the present specification, wherein the two single domain antibodies can bind to different antigen epitopes on the ROR1 protein.

[0048] An "epitope," also called an "antigenic determinant," refers to a site on an antigen that binds to a corresponding antibody molecule. An epitope may be a sequence epitope or a conformational epitope. A sequence epitope is composed of consecutively arranged amino acid residues. A conformational epitope contains amino acid residues that are not consecutively arranged, but are spatially close to each other so that they can form a specific conformation. For example, in a polypeptide, these are amino acid residues that are not adjacent in the primary sequence of the polypeptide but are sufficiently close to each other in the tertiary or quaternary structure of the polypeptide to be recognized and bound by a corresponding antibody.

[0049] "Fc fragment" refers to the handle region of a "Y"-shaped antibody molecule; i.e., the crystallizable fragment (Fc) contains the second and third constant domains (CH2 and CH3 domains) of the heavy chain. An antibody Fc region can be obtained by hydrolyzing an antibody molecule with a protease (e.g., papain). In some instances, the Fc region may contain a hinge, CH2, and CH3. When the Fc region contains the hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be derived from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" further includes variant Fc fragments derived from a native Fc fragment and modified to retain their effector function. "Variant Fc fragments" contain an amino acid sequence with at least one amino acid variation in the amino acid sequence of a native Fc fragment. In some examples, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acid substitutions in the parent Fc fragment, preferably about 1 to about 5 amino acid substitutions. In some examples, the variant Fc fragment has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parent Fc fragment. The effector function of the "Fc fragment" may include binding to an Fc receptor, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediation of phagocytosis, etc.

[0050] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence similarity") refers to the degree of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, before calculating the percent identity between two amino acid or nucleotide sequences, sequence alignment is first performed and gaps, if any, are introduced. If the amino acid residue or base in the two sequences is the same at a certain alignment position, the two sequences are considered identical or matched at that position; if the amino acid residue or base in the two sequences is different, the two sequences are considered non-identical or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matching positions by the total number of positions within the alignment window. In some other algorithms, the number and / or length of gaps are also taken into account. For the purposes of the present invention, optimal sequence alignment and sequence identity between two amino acid or nucleotide sequences can be calculated using the public alignment software BLAST (available at the website ncbi.nlm.nih.gov) with default settings. In some embodiments, "at least 90% sequence identity" as used herein includes, but is not limited to, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

[0051] In the context of an antibody or antigen-binding fragment thereof, "targeting," "toward," or "specifically binding" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., an antigen) than other molecules that are simultaneously present in the environment. A molecule can target, be directed against, or specifically bind to more than one molecule; for example, a bispecific antibody may have a higher binding affinity for two different antigens than other molecules. Several parameters, such as the EC of antibody-antigen binding, can be measured. 50The binding affinity of an antibody to an antigen can be measured by the KD value or KD value.

[0052] EC 50 EC (concentration for 50% of maximal effect) refers to the concentration that causes 50% of the maximal effect. When used to express the binding ability of antibody molecules to their corresponding antigens in enzyme-linked immunosorbent assays (ELISAs), it can refer to the antibody molecule concentration that produces half of the maximum detectable signal (e.g., colorimetric intensity or fluorescent intensity). 50 The lower the value, the higher the binding affinity to the antigen.

[0053] The KD value may be used to measure the binding affinity between an antibody and its antigen. The KD value is the equilibrium dissociation constant, or k off / k on Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.

[0054] The terms "polypeptide" and "protein" may be used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain naturally occurring or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as fucosylation, sialylation, acetylation, phosphorylation, and similar modifications. For purposes of the present invention, "polypeptide" refers to a protein, including modifications to the native sequence, such as deletions, additions, and substitutions (usually conservative in nature), so long as the protein retains the desired activity. These modifications may be deliberate, e.g., induced by site-directed mutagenesis, or may be accidental, such as mutations in the host producing the protein or errors due to PCR amplification.

[0055] As used herein, the term "variant" when referring to an antibody or antigen-binding fragment thereof refers to a protein obtained after introducing one or more amino acid insertions, deletions, or substitutions based on a parent antibody molecule, which still retains at least some of the functions of the parent antibody molecule (particularly functions of interest, such as the ability to bind to the corresponding antigen). For example, a variant of an antibody molecule may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the antigen-binding ability of the parent antibody molecule, or may even have a higher binding ability than the parent antibody molecule. In some embodiments, a variant of an antibody molecule may retain at least 80%, 85%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody molecule. In the case of an antibody molecule or antigen-binding fragment thereof, the variant typically contains amino acid modifications in the variable region framework sequence and / or constant region, although this does not exclude the possibility of making one or more amino acid modifications to the CDR region sequence. Therefore, as will be understood by those skilled in the art, by substituting, deleting, or adding some amino acids based on the specific antibody sequences of the present specification and verifying or screening the binding ability or biological activity of the resulting products with the corresponding antigen (ROR1 protein), corresponding mutants of the anti-ROR1 protein antibody molecule of the present invention can be obtained, and these mutants are also within the scope of the present invention.

[0056] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered membrane protein receptor molecule that can confer desired specificity, e.g., the ability to bind to a specific tumor antigen, to immune effector cells. A chimeric antigen receptor typically consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen-binding domain is a segment of an scFv sequence or a single-domain antibody fragment, responsible for recognizing and binding to a specific antigen. The intracellular signaling domain typically contains an immunoreceptor tyrosine-based activation motif (ITAM), e.g., a signaling domain derived from the CD3ζ molecule, and is responsible for activating and killing immune effector cells. A chimeric antigen receptor may also contain a signal peptide at the amino terminus responsible for intracellular localization of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may further contain a costimulatory domain, e.g., derived from the 4-1BB or CD28 molecule.

[0057] As used herein, "CAR cells" refer to cells that express a CAR molecule on the cell surface. In most cases, the cells are immune cells, such as T cells or NK cells. Accordingly, CAR-expressing T cells are referred to herein as "CAR-T" or "CAR-T cells." Furthermore, as used herein, unless otherwise specified, reference to CAR-T cells refers not only to cells directly modified with a CAR, but also to daughter cells produced by these cells after expansion in vitro or in vivo.

[0058] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0059] The term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, a plasmid, or a virus) that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or that can replicate in a host cell. A vector may contain one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., a promoter and / or enhancer) that control the expression of the polynucleotide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactose). The term "expression vector" refers to a vector for expressing a polypeptide of interest in a host cell.

[0060] A "host cell" refers to a cell that can be or was the isolated recipient of a vector or polynucleotide. A host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate cells, fungal cells, e.g., yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and cells derived therefrom, e.g., 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. In some embodiments, a single domain antibody according to the present disclosure may be secreted from a mammalian cell. A host cell includes the progeny of a single host cell; the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell may be an isolated cell or cell line, and also includes cells transfected in vivo with a nucleic acid molecule or expression vector according to the present disclosure. In one specific example, the host cell is a CAR cell, e.g., a CAR-T cell.

[0061] A "subject" includes animals, such as mammals, including, but not limited to, primates, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. A subject may be male or female and of any appropriate age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, a subject refers to an individual in need of diagnosis or treatment of a disease or disorder. In some examples, a subject receiving diagnosis or treatment may be a patient, which has a disorder associated with the diagnosis or treatment or is at risk of developing the disorder. In certain examples, a subject is a human, e.g., a human patient. The terms may generally be used interchangeably with "patient," "subject to be detected," "subject to be treated," etc.

[0062] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc. that can be safely administered, and which are suitable for administration to humans and / or animals without undue side effects, while maintaining the activity of the drug or active agent therein.

[0063] When referring to disease treatment, an "effective amount" refers to the amount of an active compound (e.g., an antibody) sufficient to elicit the biological or medical response desired by the clinician in a subject. The "effective amount" of an antibody administered herein can be determined by one of skill in the art based on factors such as the route of administration, the subject's weight, age, and medical condition. For example, a typical daily dose range may range from 0.01 mg to 100 mg of active ingredient per kg of body weight. Methods of administration of the active compounds (e.g., antibodies, fusion proteins) or immune effector cells provided herein include, but are not limited to, injection, e.g., intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, etc.

[0064] Antibody or antigen-binding fragment thereof targeting ROR1 protein The present specification provides an antibody or antigen-binding fragment thereof that specifically binds to the ROR1 protein. The antibody or antigen-binding fragment thereof binds to the ROR1 protein (or its Frizzled domain or Kringle domain) with relatively high binding affinity. For example, the binding ability of the antibody or antigen-binding fragment thereof to the ROR1 protein (or its Frizzled domain or Kringle domain) described in the Examples below can be measured by assay methods such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR). It can also be measured by other protein interaction assay methods known in the art, such as biolayer interferometry (BLI).

[0065] In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is obtained by screening an alpaca natural library (single domain antibody phage display library) with the ROR1 protein (or its Frizzled domain or Kringle domain). In some other embodiments, after the antibody sequence is determined, the single domain antibody is obtained by genetic engineering techniques, for example, by introducing an expression vector expressing the antibody or an antigen-binding fragment thereof into a host cell and culturing the host cell.

[0066] The present specification provides heavy chain CDR sequences for ROR1 protein (or its Frizzled domain or Kringle domain) targeting antibodies, which are as set forth in SEQ ID NOs: 2-4, 7-9, 12-14, 17-19, 22-24, 27-29, 32-34, or 37-39, respectively.

[0067] Based on these CDR sequences provided herein, those skilled in the art can construct various polypeptide constructs (including antibodies or antigen-binding fragments thereof) capable of binding to the ROR1 protein (or its frizzled domain or Kringle domain), by combining framework regions (FRs) and / or constant regions from different antibody molecules with these CDR sequences. These framework regions include natural framework region sequences from human antibodies or animal (e.g., mouse, rat, sheep, camel, etc.) antibodies. These framework regions may also include framework region sequence variants produced by modifying the natural framework region sequences. By combining the CDR sequences provided herein with different framework region sequences to form heavy chain variable regions and detecting their binding ability to the ROR1 protein (or its frizzled domain or Kringle domain), polypeptide constructs that specifically bind to the ROR1 protein (or its frizzled domain or Kringle domain) can be easily obtained.

[0068] In some embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof targeting a ROR1 protein (or its Frizzled domain or Kringle domain) according to the present specification comprises an amino acid sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31 or 36.

[0069] In some embodiments, the present specification further provides humanized antibodies of the above-mentioned single domain antibodies, which are substantially the same as the above-mentioned single domain antibodies in terms of CDR sequences, but in which alpaca antibody framework regions have been replaced with human antibody framework regions.

[0070] In some embodiments, the heavy chain variable region of a humanized antibody or antigen-binding fragment thereof targeting a ROR1 protein (or its frizzled domain or Kringle domain) according to the present specification comprises an amino acid sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:41, 43 or 45.

[0071] Based on the above single domain antibodies (including humanized single domain antibodies), the present inventors further performed affinity maturation mutations and screened some clones with improved affinity. Accordingly, in some embodiments, the inventors provide affinity matured antibodies, whose CDR sequences are as follows: (1) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:66, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:67; (2) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:66, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:69; (3) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:66, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:71; and (4) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:66, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: (5) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:75, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19; (6) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:78, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19; (7) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:80, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19; (8) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:82, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:83, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:(9) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:85, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:76, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19; (10) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:85, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:87, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19; (11) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO:89, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO:90, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO:19.

[0072] In some embodiments, the heavy chain variable region of an affinity matured antibody targeting a ROR1 protein (or its frizzled domain or Kringle domain) according to the present specification comprises an amino acid sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:65, 68, 70, 72, 73, 74, 77, 79, 81, 84, 86 or 88.

[0073] As will be understood by those skilled in the art, by substituting, deleting, or adding some amino acids based on the specific sequences herein, and verifying or screening the binding ability or biological activity of the resulting products to the ROR1 protein (or its Frizzled domain or Kringle domain), corresponding mutants of the antibody molecule targeting the ROR1 protein (or its Frizzled domain or Kringle domain) herein can be obtained, and these mutants are also included within the scope of the present invention. For example, the antibody molecule herein may have one to ten, for example, five, four, three, two, or one amino acid modifications in its full length or variable region sequence or CDR sequence. For example, the heavy chain variable region sequence shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31 or 36 may have one to ten, for example, five, four, three, two or one, amino acid modifications; the CDR sequence of SEQ ID NO: 2-4, 7-9, 12-14, 17-19, 22-24, 27-29, 32-34 or 37-39, or an affinity matured antibody may have a total of five, four, three, two or one or less amino acid modifications; and the antibody may have any combination of these modifications.

[0074] It is contemplated that the antibodies or antigen-binding fragments thereof described herein may contain conservative amino acid substitutions. Conservative amino acid substitutions may generally be described as the replacement of one amino acid residue with another amino acid residue of a similar chemical structure and have little or no substantial effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may, for example, replace one amino acid in the following groups (a) to (e) with another amino acid in the same group: (a) small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) positively charged polar residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0075] In some embodiments, an antibody or antigen-binding fragment thereof provided herein may further comprise a post-translational modification. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, fucosylation, carbonylation, ubiquitination, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, the modified soluble polypeptide may also comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. One preferred form of fucosylation is sialylation, which attaches one or more sialic acid groups to the polypeptide. The sialic acid groups improve the solubility and serum half-life of the protein and reduce the protein's potential immunogenicity.

[0076] Fusion proteins The present specification provides a fusion protein comprising at least one antibody or antigen-binding fragment thereof that specifically binds to a ROR1 protein (or its Frizzled domain or Kringle domain) according to the present specification and at least one other functional moiety. Examples of the fusion protein include, but are not limited to, a multispecific antibody (e.g., a bispecific antibody) and a chimeric antigen receptor (CAR).

[0077] In some embodiments, the antibody or antigen-binding fragment thereof can be linked to an Fc fragment to form a fusion protein. The Fc fragment can be located at the C-terminus or N-terminus of the antibody or antigen-binding fragment thereof. Preferably, the Fc fragment can be located at the C-terminus of the antibody or antigen-binding fragment thereof. The fusion protein of the antibody or antigen-binding fragment thereof formed with the Fc fragment retains the ability to specifically bind to the ROR1 protein (or its Frizzled domain or Kringle domain) and the effector functions of the Fc fragment, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. Furthermore, fusion with an Fc fragment can increase the in vivo half-life of the antibody or antigen-binding fragment thereof, thereby extending the administration interval when the antibody or antigen-binding fragment thereof is used as a therapeutic drug.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof can be linked to a protein tag to form a fusion protein. The protein tag may include a purification tag and a detectable tag. Purification tags include, but are not limited to, His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, etc. The detectable tag can be used to indicate the presence or content of ROR1 protein (or its Frizzled domain or Kringle domain) in a sample or to track the location of ROR1 protein (or its Frizzled domain or Kringle domain) in a subject's body or cells. Examples of detectable tags include various enzymes that can be used in immunodetection, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), and fluorescent proteins, such as GFP. Due to the ability of the antibody or its antigen-binding fragment to specifically bind to the ROR1 protein (or its Frizzled domain or Kringle domain), the amount of the detectable tag linked to the antibody or its antigen-binding fragment can be used to determine the amount of the antibody or its antigen-binding fragment, and further to determine the content of the ROR1 protein (or its Frizzled domain or Kringle domain) in the sample.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof can be linked to a cytokine or therapeutic protein to form a fusion protein, in which case the ability of the antibody or antigen-binding fragment thereof to specifically bind to the ROR1 protein (or its Frizzled domain or Kringle domain) allows the cytokine or therapeutic protein to be targeted to specific tissues or cells (e.g., tumor tissues expressing the ROR1 protein (or its Frizzled domain or Kringle domain)) to achieve the therapeutic effect of the cytokine or therapeutic protein.

[0080] In some embodiments, the fusion protein is a bispecific antibody, in which one antigen-binding moiety targets the ROR1 protein (or its Frizzled domain or Kringle domain) (e.g., derived from a single-domain antibody in accordance with the present disclosure), and another antigen-binding moiety can bind to a second antigen or protein other than the ROR1 protein (or its Frizzled domain or Kringle domain). In some other embodiments, the fusion protein is a bispecific antibody, in which one antigen-binding moiety targets the ROR1 protein (or its Frizzled domain or Kringle domain) and another antigen-binding moiety also targets the ROR1 protein (or its Frizzled domain or Kringle domain) (both derived from a single-domain antibody in accordance with the present disclosure), but the two antigen-binding moieties differ in amino acid sequence (particularly CDR sequence) and each binds to a different antigenic epitope on the ROR1 protein (or its Frizzled domain or Kringle domain). In one specific embodiment, one antigen-binding moiety targets the Frizzled domain and another antigen-binding moiety targets a site other than the Frizzled domain in the ROR1 protein; in another specific embodiment, one antigen-binding moiety targets the Kringle domain and another antigen-binding moiety targets a site other than the Kringle domain in the ROR1 protein; in another specific embodiment, one antigen-binding moiety targets the Kringle domain and another antigen-binding moiety targets the Frizzled domain.

[0081] In some embodiments, the two antigen-binding moieties in the bispecific antibody are linked in tandem via a peptide linker (or linker sequence). The peptide linker may be a naturally occurring linker, a synthetic linker, or a combination of both. Particularly suitable linker sequences comprise amino acid residues primarily selected from glycine (Gly), serine (Ser), alanine (Ala), and threonine (Thr). For example, the linker may contain at least 75% (calculated based on the total number of residues present in the peptide linker) (e.g., at least 80%, at least 85%, or at least 90%) of amino acid residues selected from Gly, Ser, Ala, and Thr. The linker may also consist of Gly, Ser, Ala, and / or Thr residues. In some examples, the linker contains 1 to 25 glycine residues, 5 to 20 glycine residues, 5 to 15 glycine residues, or 8 to 12 glycine residues. In some embodiments, suitable peptide linkers typically contain at least 50% glycine residues, e.g., at least 75% glycine residues. In some embodiments, the peptide linker contains only glycine residues. In some embodiments, the peptide linker contains only glycine and serine residues, for example, (GS) n where n is, for example, an integer of 1 to 20. A fusion protein formed by linking two antigen-binding moieties via a peptide linker can be used in the extracellular antigen-binding structure of a chimeric antigen receptor to construct a bispecific chimeric antigen receptor.

[0082] In some embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen is associated with enhancing or suppressing a signaling pathway in an immune cell. In some embodiments, the second antigen is a component of the T cell receptor complex, e.g., a T cell surface molecule such as CD3 (including the gamma, delta, epsilon, zeta, and eta chains).

[0083] Chimeric antigen receptor (CAR) As used herein, the CAR may comprise an extracellular antigen-binding domain that specifically binds to a ROR1 protein (or its Frizzled domain or Kringle domain), a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain.

[0084] In some embodiments, the extracellular antigen-binding domain of the CAR may comprise a single-domain antibody (or antigen-binding fragment thereof) according to the present specification. The single-domain antibody (or antigen-binding fragment thereof) can be linked to the transmembrane domain via a hinge region, for example, a CD8α hinge. The CAR can transduce immune effector cells (e.g., T cells) and be expressed on the cell surface. Thus, the present specification also provides T cells expressing the chimeric antigen receptor, and uses of the T cells and / or the CAR in drugs for treating diseases associated with ROR1 proteinopathy.

[0085] In some embodiments, the extracellular antigen-binding domain may comprise two or more single domain antibodies (or antigen-binding fragments thereof) targeting the ROR1 protein (or its Frizzled domain or Kringle domain) according to the present invention. These single domain antibodies (or antigen-binding fragments thereof) are linked in tandem, either directly or via a peptide linker. Preferably, each of these single domain antibodies (or antigen-binding fragments thereof) targets a different antigen epitope on the ROR1 protein. For example, one single domain antibody (or antigen-binding fragment thereof) targets the Frizzled domain and another single domain antibody (or antigen-binding fragment thereof) targets a site other than the Frizzled domain in the ROR1 protein; one single domain antibody (or antigen-binding fragment thereof) targets the Kringle domain and another single domain antibody (or antigen-binding fragment thereof) targets a site other than the Kringle domain in the ROR1 protein; or one single domain antibody (or antigen-binding fragment thereof) targets the Kringle domain and another single domain antibody (or antigen-binding fragment thereof) targets the Frizzled domain.

[0086] A CAR according to the present specification may comprise a transmembrane domain, which may comprise a polypeptide selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ξ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 proteins. In one specific example, the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO: 51 or a functional variant thereof.

[0087] The CAR according to the present specification may comprise a costimulatory domain, which may comprise a polypeptide selected from the proteins CD28, 4-1BB, OX40, and ICOS. In one specific example, the costimulatory domain may comprise the amino acid sequence set forth in SEQ ID NO: 52 or a functional variant thereof.

[0088] A CAR according to the present specification can comprise an intracellular signaling domain, which can comprise a signaling domain from CD3ζ. In one specific example, the intracellular signaling domain can comprise the amino acid sequence set forth in SEQ ID NO: 53 or a functional variant thereof.

[0089] The CAR according to the present specification may comprise a hinge region, which can link the transmembrane domain with an extracellular antigen-binding domain that specifically binds to the ROR1 protein (or its Frizzled or Kringle domain). In one specific example, the hinge region may comprise the amino acid sequence set forth in SEQ ID NO: 50 or a functional variant thereof.

[0090] The CAR according to the present specification may comprise a signal peptide, which may be located at the N-terminus of an extracellular antigen-binding domain that specifically binds to, for example, the ROR1 protein (or its Frizzled or Kringle domain). The signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 48 or a functional variant thereof.

[0091] The CAR according to the present specification may also be linked to a truncated peptide. In one embodiment, the truncated peptide may comprise an amino acid sequence from a T2A peptide. In one specific example, the truncated peptide may comprise the amino acid sequence set forth in SEQ ID NO: 54 or a functional variant thereof.

[0092] In some embodiments, the CAR can be further linked to a tEGFR fragment via a truncation peptide, and the tEGFR fragment can be used for signal detection (e.g., identifying CAR-positive cells) or as a molecular switch for CAR-T cells. In one specific example, the tEGFR fragment comprises the amino acid sequence set forth in SEQ ID NO: 55 or a functional variant thereof.

[0093] In some specific embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 56-63. It should be understood that these listed sequences further include a signal peptide sequence, a tEGFR fragment linked via a cleavage peptide, and these portions are not functional portions of the CAR and do not exist in the form of a fusion protein with the remaining portions after expression in T cells. Thus, in some other specific embodiments, a CAR according to the present specification comprises the remaining sequence of the amino acid sequence set forth in any one of SEQ ID NOs: 56-63 after removal of the signal peptide and / or the tEGFR fragment linked via the cleavage peptide.

[0094] Nucleic acids, vectors, cells and pharmaceutical compositions The present specification provides isolated nucleic acid molecules, which can encode the antibodies or antigen-binding fragments thereof, fusion proteins, or CARs described above. In some embodiments, the isolated nucleic acid molecules can comprise any one of the nucleic acid sequences set forth in SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44, and 46, or functional variants thereof. These nucleic acid molecules can be produced or synthesized by the following methods: (i) in vitro amplification, e.g., polymerase chain reaction (PCR), (ii) through-cloning recombinant production, (iii) purified and isolated, e.g., by enzymatic cleavage and gel electrophoresis fractionation, or (iv) synthetically synthesized, e.g., chemically synthesized. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA technology.

[0095] The present specification also provides a vector, which may contain the above-described nucleic acid molecule. The vector may be selected from one or more of a plasmid, a retroviral vector, and a lentiviral vector. In some embodiments, the nucleic acid molecule encoding a CAR is placed on a lentiviral expression vector and used to produce CAR-T cells. For example, the lentiviral vector may contain the nucleic acid sequence set forth in any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44, and 46, or a functional variant thereof. As used herein, "functional variant" refers to a nucleotide sequence that encodes the same amino acid sequence due to codon degeneracy. The vector may also contain other genes, such as marker genes, that allow for selection of the vector in an appropriate host cell under appropriate conditions. The vector may further contain expression control elements that allow for accurate expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In another aspect, the present application provides an immune effector cell, which may comprise a CAR described herein, a nucleic acid molecule described herein, or a vector described herein. In the present application, the immune effector cell may be a mammalian cell. In the present application, the immune effector cell may be selected from a T lymphocyte and a natural killer (NK) cell.

[0096] The present specification provides a method for producing immune effector cells, which includes introducing a vector described herein into the immune effector cells. For example, a vector described herein may be introduced into immune effector cells, such as T lymphocytes or natural killer (NK) cells. In some embodiments, each type or each cell may contain one or more types of vectors. In some embodiments, each type or each cell may contain multiple (e.g., two or more) or multiple types (e.g., two or more) of vectors. The vector can be introduced into immune effector cells by methods known in the art. For example, immune effector cells can be transfected with a retroviral vector to integrate the viral genome carrying a CAR molecule into the host genome, ensuring long-term and stable expression of a gene of interest. For example, a transposon can be used to introduce a plasmid carrying a CAR (transposon) and a plasmid carrying a transposase into target cells. For example, a CAR molecule can be added to the genome using gene editing techniques (e.g., CRISPR / Cas9). In the present application, the vector carrying the CAR molecule described in the present application can be introduced into the cells by methods known in the art, such as electroporation, liposome transfection, etc.

[0097] In another aspect, the present specification provides pharmaceutical compositions, which may include the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, vector, or immune effector cell, and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient may include a buffering agent, antioxidant, preservative, low-molecular-weight polypeptide, protein, hydrophilic polymer, amino acid, sugar, chelating agent, counterion, metal complex, and / or non-ionic surfactant, etc. In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration (e.g., intravenous injection, IV), intramuscular administration (e.g., intramuscular injection, IM), in situ administration at a tumor site, inhalation, rectal administration, transdermal administration, or subcutaneous depot administration.

[0098] Pharmaceutical uses The present specification provides a use of the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, vector, or immune effector cell for manufacturing a medicament for treating a disease or disorder associated with expression of ROR1. In some embodiments, the disease or disorder associated with expression of ROR1 may be tumor or cancer.

[0099] The present specification further provides a method of treating a disease, comprising administering the above-described antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, vector, or immune effector cell to a subject having or suspected of having a disease or disorder associated with expression of ROR1.

[0100] In some embodiments, the disease or disorder associated with expression of ROR1 is a tumor or cancer.

[0101] In some embodiments, the tumor comprises a solid tumor such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and ovarian cancer.

[0102] Tumor immunotherapy by targeting ROR1 is an extremely useful therapeutic method, and the inventors have developed new functional monoclonal nanoantibodies (single-domain antibodies) and related chimeric antigen receptor T cell therapies that target human ROR1 and have specific multiple antigen-recognition epitopes, thereby reducing the possibility of drug resistance by specifically binding to different ROR1 antigen-binding epitopes, suppressing the growth of ROR1-positive tumor cells, and treating cancer diseases that express ROR1.

[0103] The present invention relates to antibodies and CAR-T molecules with functional targets against human ROR1, and the following describes in detail the embodiments of the present invention in conjunction with examples. Unless otherwise specified, the technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the methods and materials used in the examples described below are all common products that are commercially available. As will be understood by those skilled in the art, the methods and materials described below are merely illustrative and should not be construed as limiting the scope of the present invention.

[0104] Example 1: Obtaining human ROR1 single domain antibody-positive clone molecules 1) Screening for lead antibody molecules that specifically target human ROR1 using an alpaca natural library Using an alpaca natural library independently developed by Hobo Biosystems, three rounds of liquid-phase or solid-phase screening were performed with human ROR1-Biotin protein (fused biotin protein tag, Acrobiosystems, RO1-H821y, sequence see Q01973-1), human ROR1 Frizzled-His domain protein (amino acid sequence of positions 165 to 305, fused His protein tag, Acrobiosystems, RO1-H5222, sequence see Q01973-1), and human ROR1 Kringle-His domain protein (amino acid sequence of positions 308 to 395, fused His protein tag, Acrobiosystems, RO1-H5223, sequence see Q01973-1), and phage library eluates were obtained.

[0105] 2) Screening for positive clones The neutralized phage panning eluate was added to the prepared TG1 bacterial suspension, mixed uniformly, and incubated at 37°C for 45 minutes to infect the TG1 host bacteria. After sufficient infection, the bacterial suspension was diluted by gradient and plated onto an agar plate with the appropriate resistance, inverted, and cultured overnight at 37°C. A 96-well sterile deep-well plate was prepared with 0.5 ml of 2YT medium (containing 0.2% w / v glucose and 0.1 mg / ml ampicillin antibiotic). Monoclonal colonies grown on the plate were picked with a sterile pipette tip and placed in the well plate. The plate was then cultured overnight (16-18 hours) at 37°C with shaking at 220 rpm. 0.05-0.1 ml of the monoclonal bacterial solution cultured overnight was transferred to a newly prepared sterile deep well plate (dispensed with 0.5 ml of 2YT medium containing ampicillin antibiotics at a final concentration of 0.1 mg / ml) and cultured until the OD value reached approximately 0.6-0.8 (under OD600 detection conditions). Helper phage was added, and the plate was shaken uniformly. After incubation at 37 ° C for 45 minutes, 0.5 ml of 2YT medium (containing 0.1 mg / ml of ampicillin antibiotics, the final concentration of which was 0.05 mg / ml after the addition of ampicillin antibiotics) was added, and the plate was cultured overnight (16-18 hours) at 25 ° C with shaking at 220 rpm. The overnight expression solution was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant. This was then used for ELISA binding detection of the monoclonals after test panning with the antigen proteins human ROR1-His protein (fused His protein tag, AcroBiosystems, RO1-H522y), human ROR1 Frizzled-His domain protein, and human ROR1 Kringle-His domain protein, and for FACS binding detection against cellular antigens to obtain positive single-domain antibody clones.

[0106] ELISA binding detection method: Indirect ELISA was used to evaluate the binding ability of phage-displayed antibodies in the supernatants to human ROR1-related proteins. ELISA microplates were coated overnight at 4°C with 1 μg / ml of recombinant human ROR1-His protein, human ROR1 Frizzled-His domain protein, and human ROR1 Kringle-His domain protein in 100 μl / well of CBS coating reagent. The plates were washed with PBS-T (0.05% Tween) and blocked with 300 μl / well of PBS containing 3% skim milk at 37°C for 1 hour. The mounting solution was then discarded, and 50 μl of phage-expressed supernatant and 50 μl of 0.1% PBST were added to each plate and incubated at room temperature for 2 hours. Plates were washed three times with PBST and incubated with 100 μl / well of goat anti-M13 phage antibody conjugated with horseradish peroxidase (Yiqiao) for 45 minutes at room temperature. Plates were washed six times with PBST, and TMB color development solution (GenScript) was added and incubated for 10-15 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl of 1 M HCl stop solution (Sigma). Plates were read at 450 nm using a microplate reader.

[0107] FACS detection method: FACS binding assay was used to evaluate the binding ability of antibodies in the supernatant to the human ROR1 antigen expressed on the membrane surface of CHO cells. CHO cells expressing human ROR1 and negative control parent cells were collected and washed three times with PBS. 2.5x10 cells were placed in a 96-well plate. 5 100 μl of the detection cells, 100 μl of the target supernatant, and 3.5 μg / ml biotin-labeled anti-phage antibody were added and incubated at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of iFluorescent-labeled streptavidin protein (Jackson, 016-600-084) was added and incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS, and the signal was read using a FACS BD Calibur.

[0108] Example 2: Variable region sequencing of positive clones and recombinant production of monoclonal antibodies Based on the ELISA and FACS detection results, positive clones were selected, and the original colonies corresponding to the positive clones were aspirated and cultured in liquid. The expression plasmids were extracted, and PCR and monoclonal Sanger sequencing were performed. Finally, eight positive clones were obtained, as shown in Table 1 below.

[0109] [Table 1]

[0110] The sequence of the antibody is as follows: AHP15485 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1 AHP15485 CDR1 region amino acid sequence SEQ ID NO: 2 AHP15485 CDR2 region amino acid sequence SEQ ID NO: 3 AHP15485 CDR3 region amino acid sequence SEQ ID NO: 4 AHP15485 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 5 AHP15547 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 6 AHP15547 CDR1 region amino acid sequence SEQ ID NO: 7 AHP15547 CDR2 region amino acid sequence SEQ ID NO: 8 AHP15547 CDR3 region amino acid sequence SEQ ID NO: 9 AHP15547 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 10 AHP15580 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 11 AHP15580 CDR1 region amino acid sequence SEQ ID NO: 12 AHP15580 CDR2 region amino acid sequence SEQ ID NO: 13 AHP15580 CDR3 region amino acid sequence SEQ ID NO: 14 AHP15580 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 15 AHP15662 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 16 AHP15662 CDR1 region amino acid sequence SEQ ID NO: 17 AHP15662 CDR2 region amino acid sequence SEQ ID NO: 18 AHP15662 CDR3 region amino acid sequence SEQ ID NO: 19 AHP15662 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 20 AHP15768 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 21 AHP15768 CDR1 region amino acid sequence SEQ ID NO: 22 AHP15768 CDR2 region amino acid sequence SEQ ID NO: 23 AHP15768 CDR3 region amino acid sequence SEQ ID NO: 24 AHP15768 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 25 AHP15773 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 26 AHP15773 CDR1 region amino acid sequence SEQ ID NO: 27 AHP15773 CDR2 region amino acid sequence SEQ ID NO: 28 AHP15773 CDR3 region amino acid sequence SEQ ID NO: 29 AHP15773 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 30 AHP15776 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 31 AHP15776 CDR1 region amino acid sequence SEQ ID NO: 32 AHP15776 CDR2 region amino acid sequence SEQ ID NO: 33 AHP15776 CDR3 region amino acid sequence SEQ ID NO: 34 AHP15776 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 35 AHP16026 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 36 AHP16026 CDR1 region amino acid sequence SEQ ID NO: 37 AHP16026 CDR2 region amino acid sequence SEQ ID NO: 38 AHP16026 CDR3 region amino acid sequence SEQ ID NO: 39 AHP16026 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 40 A DNA fragment containing the codon-optimized heavy chain variable region was synthesized and inserted into the pcDNA3.4-Fc(HuIgG1) expression vector to form an expression plasmid.

[0111] HEK293-6E cells were transfected with the above plasmids and cultured in shake flasks at 37°C for 10 days. The supernatant was then harvested and used for antibody purification. Prior to purification, the tubes and Protein A column were depyrogenated with 0.2 M NaOH. The column was re-equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0). The resulting cell culture supernatant was then diluted 1:1 with 2x the above buffer and sterilized by filtration. The filtered supernatant and Protein A column were incubated at room temperature for 2 hours. After washing the column with 1x the above buffer, the IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1 M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was replaced with PBS (pH 7.4), the elution buffer was removed, and the sample was concentrated. After concentration, the antibody was quantified by OD280nm using an absorption coefficient Ec of 1.43 (0.1%).

[0112] Purified antibodies were analyzed by SDS-PAGE on 10% precast gels (GenScript) using a BioRad electrophoresis system. The gels were stained with Estain 2.0 (GenScript), and molecular size and purity were estimated by comparing the stained bands with the Protein Ladder (GenScript).

[0113] Example 3: ELISA binding detection of monoclonal antibodies with human ROR1 recombinant protein Indirect ELISA was performed to evaluate the binding ability of the purified antibodies to human ROR1-related recombinant proteins. ELISA plates (Nunc) were coated overnight at 4°C with 100 μl / well of 1 μg / ml human ROR1-His protein, human ROR1 Frizzled-His domain protein, and human ROR1 Kringle-His domain protein in CBS. The plates were washed with PBS-T (0.05% Tween) and blocked with 300 μl / well of PBS containing 3% skim milk at 37°C for 1 hour. The mounting solution was then discarded, and 100 μl of 8 μg / ml (approximately 100 μm) purified antibody was added to the first well and diluted three-fold to achieve a total of 11 test concentration gradients. The plates were then incubated at room temperature for 1 hour. The plate was washed three times with PBST and incubated with 100 μl / well of mouse anti-human IgG Fc fragment conjugated with horseradish peroxidase (GenScript) for 0.5 hours at 37°C. The plate was washed five times with PBST, and TMB color development solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. Figures 1-3 show the ELISA experiment results for the binding of eight positive recombinant antibody clones to ROR1-related antigen proteins, and the EC values ​​of each antibody. 50As shown in Table 2 below, all of these test antibodies reached or exceeded the antigen-binding capacity of the positive control antibody R11-scFv.

[0114] [Table 2]

[0115] Example 4: Binding of monoclonal antibodies to cell lines expressing human and mouse ROR1 CHO cells expressing human and mouse ROR1 and negative control parental cells were collected and washed three times with PBS. 2.5x10 cells were plated in a 96-well plate. 5 100 μl of 10 μg / ml purified antibody was added to the detection cells and incubated for 1 hour at 4°C. The cells were then washed three times with PBS, and 100 μl of iFluorescent-labeled goat anti-human IgG, Fcγ-specific fragment antibody was added and incubated for 45 minutes at 4°C. Finally, the cells were washed three times with PBS, and signals were read using a FACS BD Calibur. As shown in Figure 4, all antibodies bound to both ROR1-expressing CHO-K1 / Human ROR1 stable cells and CHO-K1 / Mouse ROR1 stable cells, but not to the parental CHO cells.

[0116] Example 5: Humanized design and recombinant production of single domain antibody molecules targeting human ROR1 The parent antibody structure was modeled using computer-aided homology modeling software (MOE). A natural human germline sequence with high homology to the parent sequence was selected. Using CDR grafting technology, the CDRs of the positive monoclonal antibody were grafted onto the natural human germline sequence to obtain a humanized chimeric antibody of the parent antibody. The different amino acid residues of the chimeric and parent antibodies were compared, and key points that could affect subsequent affinity were identified based on classical residues, interaction loop regions, core regions, and mutation hot spots. Appropriate sites were selected for combinatorial design to obtain the humanized antibody variable region amino acid sequence. The humanized antibody amino acid sequence was codon-optimized, and a DNA fragment containing the codon-optimized heavy chain variable region was synthesized and inserted into the pcDNA3.4-Fc (Human IgG1) expression vector to form an expression plasmid. The relevant expression plasmid was produced according to the antibody recombinant expression and purification methods described in Example 2, and the humanized antibody was obtained.

[0117] AHP15485-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 41 AHP15485-VHH4 CDR1 region amino acid sequence SEQ ID NO: 2 AHP15485-VHH4 CDR2 region amino acid sequence SEQ ID NO: 3 AHP15485-VHH4 CDR3 region amino acid sequence SEQ ID NO: 4 AHP15485-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 42 AHP15662-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 43 AHP15662-VHH4 CDR1 region amino acid sequence SEQ ID NO: 17 AHP15662-VHH4 CDR2 region amino acid sequence SEQ ID NO: 18 AHP15662-VHH4 CDR3 region amino acid sequence SEQ ID NO: 19 AHP15662-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 44 AHP15773-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 45 AHP15773-VHH4 CDR1 region amino acid sequence SEQ ID NO: 27 AHP15773-VHH4 CDR2 region amino acid sequence SEQ ID NO: 28 AHP15773-VHH4 CDR3 region amino acid sequence SEQ ID NO: 29 AHP15773-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 46

[0118] Example 6: SPR binding detection of humanized antibodies to human ROR1-related proteins The affinity of purified antibodies to human ROR1-associated proteins was measured independently using a surface plasmon resonance (SPR) biosensor, Biacore T200 (GE Healthcare). Antibodies were immobilized on the sensor chip using the Fc capture method. Human ROR1-associated proteins were used as analytes. Data on dissociation (kd) and association (ka) rate constants were obtained using the Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated as the ratio of kd to ka. Antibodies were ranked according to their equilibrium dissociation constants, and humanized antibodies with no or less than three-fold decrease in affinity were selected for further detection. The SPR affinity measurement results are shown in Table 3 below, and the associated sensor diagram is shown in Figure 5. SPR affinity measurements revealed that the parent and humanized antibodies in the three groups all had similar affinity levels with the human ROR1 antigen protein, with equilibrium dissociation constants (KD) of 10 or greater. -7 Within range.

[0119] [Table 3]

[0120] Example 7: Binding of humanized antibodies to cell lines expressing human and mouse ROR1 CHO cells expressing human and mouse ROR1 and negative control parental cells were collected and washed three times with PBS. 2.5x10 cells were plated in a 96-well plate. 5 100 μl of 10 μg / ml purified antibody was added to the detection cells and incubated for 1 hour at 4°C. The cells were then washed three times with PBS, and 100 μl of iFluorescent-labeled goat anti-human IgG, Fcγ-specific fragment antibody was added and incubated for 45 minutes at 4°C. Finally, the cells were washed three times with PBS, and the signals were read on a FACS BD Calibur. As shown in Figure 6, all humanized antibodies bound to both ROR1-expressing CHO-K1 / Human ROR1 stable cells and CHO-K1 / Mouse ROR1 stable cells, but not to the parental CHO cells, comparable to their corresponding parental samples.

[0121] Example 8: Construction and detection of human ROR1 modified cell lines After synthesizing the ROR1 DNA ORF sequence (the sequence is derived from NP_005003.2, and the amino acid sequence is as shown in SEQ ID NO:47), the DNA fragment of ROR1 ORF was ligated with the vector backbone pLVX-Puromycin by Clone EZ (GenScript) technology and transformed into E. coli competent cells to obtain the plasmid pLVX-ROR1-puromycin.

[0122] Lentivirus packaging: Using the triplasmid system, pLVX-ROR1-puromycin and two helper vector plasmids (psPAX2, PMD2.G) were co-transfected into 293T cells. The 293T cell supernatant was collected and concentrated and purified by ultracentrifugation. The collected virus was stored at -80°C.

[0123] Cell infection: CHO-K1 cells (ECACC) were seeded in a 6-well plate, 3 mL of medium was added, and the cells were cultured overnight. Before infection, the cells were removed from the refrigerator and the virus was quickly thawed in a 37°C water bath. The original medium was aspirated, and 1 / 2 volume of fresh medium was added. The virus stock solution was then added to the cells and mixed evenly. After 0.5-1 hour of centrifugation and infection, the culture plate was transferred to a 37°C, 5% CO2 incubator and cultured for 24 hours. On the second day (approximately 24 hours) after infection, the virus-containing medium was aspirated, and fresh complete medium was added. The cells were then continued to be cultured at 37°C.

[0124] Puromycin resistance screening: 8 μg / ml puromycin (Thermo Fisher Scientific, Cat. #A11138-03) was added to the cell culture medium, and the complete culture medium containing puromycin was replaced every 2–3 days until the uninfected control cells completely died under the screening pressure of puromycin. Continuous screening was performed until a stable cell line was obtained.

[0125] FACS detection of ROR1 expression: A portion of the obtained stable cells was taken, placed in a FACS tube, and centrifuged to remove the supernatant. PE anti-human ROR1 antibody (BioLegend, Cat#357804) was added, and the cells were incubated at 4°C for 30 minutes. After 30 minutes, the cells were washed to remove the supernatant, resuspended in FACS buffer, and analyzed on a machine (BD FACS Celesta TM ) to detect ROR1 expression levels.

[0126] Monoclonal selection: The cell pool was diluted to the limit and placed in a 96-well plate. After 7 days, the 96-well plate was examined under a microscope and wells containing monoclonal cells were marked. The monoclonal cells were then transferred to a 24-well plate and subsequently expanded to a 6-well plate. After monoclonal expansion, the expression levels of the CHO-K1 / ROR1 monoclonals were retested using the above steps. The optimal clones were shown in Figure 7, where FACS detected CHO-K1 / ROR1 overexpression.

[0127] Example 9: CAR vector construction The present invention constructed a second-generation CAR vector targeting human ROR1, whose structure is shown in Figure 8 and sequentially contains, for example, a ROR1 VHH antibody sequence, a CD28 transmembrane domain, a 4-1BB intracellular domain sequence, a CD3ζ sequence, and a tEGFR sequence as a transduction indicator protein. The amino acid sequences of the CARs of the present invention (AHP15485 CAR, AHP15580 CAR, AHP15662 CAR, AHP15773 CAR, AHP15768 CAR, AHP16026 CAR, AHP15547 CAR, and AHP15776 CAR) and R12 CAR are shown in SEQ ID NOs: 56 to 64.

[0128] Example 10: Construction of targeted human ROR1-CAR-Jurkat cells To preliminarily verify the functional effect of ROR1 CAR, ROR1-CAR-Jurkat cells were constructed.

[0129] Jurkat / NFAT-Luc cells were constructed. The construction steps were as follows: Jurkat cells (ATCC) were cultured at 3 x 10 5 The cells were seeded onto a 6-well plate, and the NFAT-Luc virus concentrate was added to the cells for infection. Three days after infection (approximately 48 hours), resistance screening was performed using Hygromycin B (Thermo Fisher, Cat# 10687-010) to obtain a Jurkat / NFAT-Luc cell pool.

[0130] ROR1 CAR concentrated virus was produced. Lentivirus was prepared according to the lentivirus packaging technique described in Example 8 of the present specification for the construction of ROR1 modified cell lines.

[0131] ROR1-CAR-Jurkat cells were prepared. A pool of Jurkat / NFAT-Luc cells was infected with concentrated ROR1 CAR virus at an MOI of 10. 72 h postinfection, CAR expression was detected by FACS. As shown in Table 4, FACS was used to detect the expression of the CAR transduction indicator protein EGFR (PE-EGFR Antibody, Novus, NBP2-75903PE). The results showed that transduction efficiencies were greater than 99%.

[0132] [Table 4]

[0133] Example 11: Targeted human ROR1-CAR-Jurkat cells are specifically activated in target cells To verify whether the ROR1-CAR constructed in this invention can be specifically activated by ROR1-positive cells, ROR1-CAR Jurkat cells (SEQ ID NO: 56-63) with eight different ROR1 VHH sequences and R12 ScFv (Patent Publication No.: WO 2014 / 031687 A1) were constructed, and the constructed R12 CAR-Jurkat cells were used as the positive control for the experiment (SEQ ID NO: 64). After co-incubation with CHO-K1 / ROR1 cells, the fluorescent signal value and IL-2 cytokine secretion were detected.

[0134] Based on the reporter gene method, the activation effect of ROR1-CAR on Jurkat cells was evaluated: target cells CHO-K1 / ROR1 were collected and resuspended in complete medium F12K (Gibco, Cat#21127-022) + 10% FBS (Gibco, Cat#10091-148) to adjust the density to 2E5 cells / mL, and 50 μL of the target cell suspension was transferred to a 96-well plate. The culture plate containing the target cells was transferred to an incubator and incubated overnight at 37°C, 5% CO2. The next day, effector cells ROR1-CAR Jurkat were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148), and the density was adjusted to 4E5 cells / mL. The overnight cultured cell culture plate was removed from the incubator, the previous medium was discarded, 100 μL of the effector cell suspension was transferred, and the culture plate was transferred to the incubator and incubated at 37°C, 5% CO2 for 6 hours. The culture plate was removed and the detection reagent Fire-LumiNova was added. TM After adding a luciferase detection kit (GenScript, Cat# L00877C) and incubating at room temperature for 5 minutes, the luminescence signal was detected using a microplate reader (PHERAStar FSX, BMG). The results are shown in Figure 10 and Table 5. Of the eight sequences, sequences AHP15485, AHP15580, AHP15662, AHP15773, and AHP15776 showed relatively high activation effects on Jurkat cells, with signal ratios closer to those of the positive control R12 ScFv.

[0135] [Table 5]

[0136] The activation effect of ROR1-CAR on Jurkat cells was evaluated based on the cytokine IL-2 secretion level: target cells CHO-K1 / ROR1 were collected and resuspended in complete medium F12K (Gibco, Cat#21127-022) + 10% FBS (Gibco, Cat#10091-148) to adjust the density to 2E5 cells / mL, and 50 μL of the target cell suspension was transferred to a 96-well plate. The culture plate containing the target cells was transferred to an incubator and incubated overnight at 37°C, 5% CO2. The next day, effector cells (ROR1-CAR Jurkat) were collected and resuspended in complete medium 1640 (Gibco, Cat# 22400-089) + 10% FBS (Gibco, Cat# 10091-148), and the density was adjusted to 4E5 cells / mL. The overnight culture plate was removed from the incubator, the previous medium was discarded, 100 μL of the effector cell suspension was transferred, and the culture plate was transferred to an incubator and incubated at 37 °C, 5% CO for 24 h. The culture supernatant was collected, and IL-2 cytokine secretion was detected using a Human IL2 kit (Cisbio, Cat# 62HIL02PEH) according to the manufacturer's instructions. The signal value was detected using a microplate reader (PHERAStar FSX, BMG). The results are shown in Figure 11 and Table 6, and indicate that the molecular sequences AHP15485, AHP15580, AHP15662, AHP15773 and AHP15776 have relatively high T cell activation effects and have signal ratios closer to those of the positive control R12 ScFv.

[0137] [Table 6]

[0138] ROR1-CAR-Jurkat cells showed different degrees of fluorescent signal values ​​and increased IL-2 secretion, indicating that the eight ROR1-CAR-Jurkat cells constructed in this invention can be specifically activated into ROR1-positive cells. This suggests that the molecular sequences AHP15485, AHP15580, AHP15662, AHP15773 and AHP15776 have a higher T cell activation effect. The fluorescent signal value detection results of the reporter gene method are consistent with the IL-2 cytokine secretion detection results.

[0139] Example 12: Construction and identification of ROR1-CAR-T cells To further verify the function of each ROR1-CAR in primary T cells, we constructed ROR1-CAR-T cells using eight different ROR1 VHH sequences via lentiviral transfection, and used R12 CAR-T cells constructed with the R12 ScFv as a positive control. The specific construction steps are as follows:

[0140] T cell activation: After resuscitation of cryopreserved PBMCs (died), the cells were resuspended in AIM V complete medium (ThermoFisher, Cat# 31035025) containing 300 U / ml rhIL-2 and counted. The T cells were then activated with CD3 / CD28 antibody magnetic beads (GenScript) at a 1:1 ratio (the activation time point was recorded as D0, and the 1st and 2nd days after activation were recorded as D1 and D2, respectively). The activated T cells were obtained after co-incubation with the magnetic beads for 24 hours.

[0141] Infection of T cells with lentivirus: Activated T cells were seeded into 24-well plates, with a cell density of 1E6 cells per well. CAR lentivirus (MOI = 10) was added to a total volume of 400 μl, along with 16 μl of the infection-promoting reagent HiTransG P (Jikai Gene, Cat# REVG005). After 16 hours of transfection, 1 ml of medium was added to each well and the cells were cultured for 72 hours to obtain ROR1-targeting CAR-T cells.

[0142] Flow cytometry detection of T cell percentage: Three days after transfection (activating D4), the percentage of CD3 was detected by FACS. The flow cytometry results are shown in Figure 9, which show that the percentage of CD3 was greater than 99% and the percentage of T cells in the cell population was greater than 99%.

[0143] Flow cytometry detection of CAR cell percentage: Three days after transfection (activating D4), CAR transduction efficiency was detected by FACS. The CAR positive rate results are shown in Table 7, and the CAR transfection efficiency was over 70% in all cases.

[0144] [Table 7]

[0145] Example 13: In vitro killing experiment of ROR1-CAR-T cells against ROR1-positive target cells Target cells CHO-K1 / ROR1 / Luc (Nanjing Probio, Cat# RD00949) were harvested and resuspended in complete medium 1640 (Gibco, Cat# 22400-089) + 10% FBS (Gibco, Cat# 10091-148), the density was adjusted to 1E5 cells / mL, and 50 μL of the target cell suspension was transferred to a 96-well plate. ROR1-CAR-T cells were collected and resuspended in complete medium 1640 (Gibco, Cat# 22400-089). The theoretical density was adjusted to 2E6 cells / mL based on the CAR-positive rate of different transduction molecules (see Table 7). 50 μL of the ROR1-CAR-T cell suspension and 50 μL of 1% Triton X-100 (Beijing Bailingwei Technology Co., Ltd., Cat# 993361) (maximum killing signal value) were transferred to the 96-well plate seeded with target cells. The culture plate was then transferred to an incubator and incubated at 37°C, 5% CO2 for 24 hours. After 24 hours of incubation, the culture plate was removed and the detection reagent Fire-LumiNova was added. TMA luciferase detection kit (GenScript, Cat#L00877C) was added, and after incubation at room temperature for 5 minutes, the luminescence signal value was detected using a microplate reader (PHERAStar FSX, BMG).

[0146] Killing formula % Cytotoxicity=100*(1-(RLU Experimental -RLU Min ) / (RLU UnT -RLU Min The ROR1-CAR-T killing values ​​of different transduction molecules were calculated using the above method. The results are shown in Figure 12 and Table 8. CAR T cells with molecular sequences AHP15485, AHP15662, and AHP15773 exhibited killing efficiencies comparable to the positive control R12 CAR T (AHP15485 % Cytotoxicity = 84.86%, AHP15662 % Cytotoxicity = 96.69%, AHP15773 % Cytotoxicity = 94.82%, R12 CAR % Cytotoxicity = 90.28%).

[0147] RLU Experimental : Signal value of ROR1-CAR T cells + target cells CHO-K1 / ROR1 / Luc, RLU UnT : Signal value of non-transduced T cells + target cells CHO-K1 / ROR1 / Luc. The target cells were not specifically killed by Car-T cells, and the signal value was the highest. RLU Min : Signal value of 1% Triton X-100 + target cells CHO-K1 / ROR1 / Luc. The target cells were completely lysed by Triton X-100, and the signal value was the lowest (RLU in the killing calculation formula). Min ).

[0148] [Table 8]

[0149] Example 14: Cytokine release experiment of ROR1-CAR-T cells against ROR1-positive target cells The target cells CHO-K1 / ROR1 / Luc were collected and resuspended with complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148) to adjust the density to 5E4 cells / mL, and 100 μL of the target cell suspension was transferred to a 96-well plate. The ROR1-CAR T cells of different transduction molecules were collected and resuspended with complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148) to adjust the theoretical density to 1E6 cells / mL based on the CAR positive rate of different transduction molecules (see Table 7), and 100 μL of the ROR1-CAR T cells were transferred to a 96-well plate. The suspension was transferred to a 96-well plate seeded with target cells, and the culture plate was transferred to an incubator and incubated at 37°C, 5% CO2 for 24 h. After 24 hours of incubation, the culture plates were removed and the culture supernatants were collected. The secretion of IL-2 and IFN-γ cytokines was detected using a Human IL2 kit (Cisbio, Cat# 62HIL02PEH) and a Human IFN gamma kit (Cisbio, Cat# 62HIFNGPEH) according to their respective manufacturer's instructions. The signal values ​​were detected using a microplate reader (PHERAStar FSX, BMG). The results are shown in Figure 13 and Tables 9 and 10. Among the eight molecular sequences, the CAR T cell cytokine secretion levels of molecular sequences AHP15485, AHP15662, and AHP15773 were higher and comparable to that of the positive control R12 CAR T. (IL-2 secretion level comparison: AHP15485 S / B ratio=109.00, AHP15662 S / B ratio=99.87, AHP15773 S / B ratio=109.00, AHP15662 S / B ratio=99.87, AHP15773 S / B ratio=109.00). ratio=107.49, R12 CAR S / B ratio=265.67, IFN-γ secretion level comparison: AHP15485 S / B ratio=46.87, AHP15662 S / B ratio=48.45, AHP15773 S / B ratio=58.03, R12 CAR S / B ratio=51.33), the detection results and killing effect were consistent.

[0150] [Table 9]

[0151] [Table 10]

[0152] Example 15: Affinity maturation and recombinant validation of humanized single domain antibodies The humanized single domain antibody was synthesized in a prokaryotic soluble expression vector from Penbo, and prokaryotic expression and binding verification were performed. After confirming normal expression, the prokaryotic soluble expression plasmid of the single domain antibody was used as a template for the subsequent mutation library. An accurate saturation mutation library of the CDR region of the single domain antibody was ordered from Jinsirui. After receiving the synthesized library liquid sample, ELISA binding detection with the antigen protein was performed. Based on the ELISA detection results, clones with improved binding values ​​were selected and subjected to SPR ranking detection. Based on the dissociation constant ratio between the sample and the humanized single domain antibody sample in the SPR binding detection results, significantly improved clones were selected and subjected to Sanger sequencing to determine the specific mutation sites and mutated amino acids of the improved clones.

[0153] Based on the mutation status of the improved clones in the precise saturation mutation library, a second round of combinatorial mutations and related library construction primers were designed, and the humanized single-domain antibody prokaryotic soluble expression plasmid was still used as a template to construct the combinatorial mutation library. The subsequent screening procedures were consistent with the precise saturation mutation library, and finally, the top five or seven clones with the highest affinity improvement fold were selected for recombination validation. For the humanized antibody AHP15485, five affinity-improved clones (AHF22016, AHF22018, AHF22013, AHP15485-VHH4-Variant1, AHP15485-VHH4-Variant2) were obtained through affinity maturation screening, which were then subjected to eukaryotic expression purification and subsequent affinity multi-concentration detection. For AHP15662-VHH4, seven clones (AHP15662-VHH4-Variant1, AHP15662-VHH4-Variant2, AHP15662-VHH4-Variant3, AHF21711, AHF21719, AHF21720, AHF21721) were obtained. After eukaryotic recombinant expression and purification of the affinity-improved clones, the affinity of the purified antibodies to human ROR1-related proteins was independently measured using a surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). For details on the sequences of the affinity-improved clones, see the sequences below. The specific affinity detection method is the same as in Example 6.

[0154] AHF22013 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 65 AHF22013 CDR1 region amino acid sequence: SEQ ID NO: 66 AHF22013 CDR2 region amino acid sequence: SEQ ID NO: 3 AHF22013 CDR3 region amino acid sequence: SEQ ID NO: 67 AHF22016 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 68 AHF22016 CDR1 region amino acid sequence: SEQ ID NO: 66 AHF22016 CDR2 region amino acid sequence: SEQ ID NO: 3 AHF22016 CDR3 region amino acid sequence: SEQ ID NO: 69 AHF22018 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 70 AHF22018 CDR1 region amino acid sequence: SEQ ID NO: 66 AHF22018 CDR2 region amino acid sequence: SEQ ID NO: 3 AHF22018 CDR3 region amino acid sequence: SEQ ID NO: 71 AHP15485-VHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 72 AHP15485-VHH4-Variant1 CDR1 region amino acid sequence: SEQ ID NO: 2 AHP15485-VHH4-Variant1 CDR2 region amino acid sequence: SEQ ID NO: 3 AHP15485-VHH4-Variant1 CDR3 region amino acid sequence: SEQ ID NO: 4 AHP15485-VHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 73 AHP15485-VHH4-Variant2 CDR1 region amino acid sequence: SEQ ID NO: 66 AHP15485-VHH4-Variant2 CDR2 region amino acid sequence: SEQ ID NO: 3 AHP15485-VHH4-Variant2 CDR3 region amino acid sequence: SEQ ID NO: 4 AHP15662-VHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 74 AHP15662-VHH4-Variant1 CDR1 region amino acid sequence: SEQ ID NO: 75 AHP15662-VHH4-Variant1 CDR2 region amino acid sequence: SEQ ID NO: 76 AHP15662-VHH4-Variant1 CDR3 region amino acid sequence: SEQ ID NO:19 AHP15662-VHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 77 AHP15662-VHH4-Variant2 CDR1 region amino acid sequence: SEQ ID NO: 78 AHP15662-VHH4-Variant2 CDR2 region amino acid sequence: SEQ ID NO: 76 AHP15662-VHH4-Variant2 CDR3 region amino acid sequence: SEQ ID NO:19 AHP15662-VHH4-Variant3 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 79 AHP15662-VHH4-Variant3 CDR1 region amino acid sequence: SEQ ID NO: 80 AHP15662-VHH4-Variant3 CDR2 region amino acid sequence: SEQ ID NO: 76 AHP15662-VHH4-Variant3 CDR3 region amino acid sequence: SEQ ID NO:19 AHF21711 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 81 AHF21711 CDR1 region amino acid sequence: SEQ ID NO: 82 AHF21711 CDR2 region amino acid sequence: SEQ ID NO: 83 AHF21711 CDR3 region amino acid sequence: SEQ ID NO:19 AHF21719 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 84 AHF21719 CDR1 region amino acid sequence: SEQ ID NO: 85 AHF21719 CDR2 region amino acid sequence: SEQ ID NO: 76 AHF21719 CDR3 region amino acid sequence: SEQ ID NO:19 AHF21720 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 86 AHF21720 CDR1 region amino acid sequence: SEQ ID NO: 85 AHF21720 CDR2 region amino acid sequence: SEQ ID NO: 87 AHF21720 CDR3 region amino acid sequence: SEQ ID NO:19 AHF21721 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 88 AHF21721 CDR1 region amino acid sequence: SEQ ID NO: 89 AHF21721 CDR2 region amino acid sequence: SEQ ID NO: 90 AHF21721 CDR3 region amino acid sequence: SEQ ID NO:19 The results are shown in Tables 11 to 12 and Figures 14 to 15. After affinity maturation, the humanized single domain antibody AHP15485-VHH4 showed an affinity of up to 10. -9 The humanized single domain antibody AHP15662-VHH4 showed an affinity of up to 10 -8 level rose to.

[0155] [Table 11]

[0156] [Table 12]

[0157] The amino acid and nucleotide sequence information referred to herein is as follows:

[0158] AHP15485 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1 EVQLVESGGGLAQAGGSLSLSCTASGRSFSNFQMGWFRQAPGKEREFVAASGWSGGPTYYADSVKGRFTISTDDSKKTMYLQMNSLKPEDTAAYYCYEDRVLSGRPVRYWGRGTQVTVSS AHP15485 CDR1 region amino acid sequence SEQ ID NO: 2 GRSFSNFQ AHP15485 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHP15485 CDR3 region amino acid sequence SEQ ID NO: 4 YEDRVLSGRPVRY AHP15485 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 5 GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGCGCAGGCGGGCGGCAGCCTGAGCCTGAGCTGCACCGCGAGCGGCCGTAGCTTTAGCAACTTTCAGATGGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTTGTGGCGGCGAGCGGCTGGAGCGGCGGCCCGACCTATTAT GCGGATAGCGTGAAAGGCCGTTTTACCATTAGCACCGATGATAGCAAAAAAACCATGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGCGTATTATTGCTATGAAGATCGTGTGCTGAGCGGCCGTCCGGTGCGTTATTGGGGCCGTGGCACCCAGGTGACCGTGAGCAGC AHP15547 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 6 AVQLVDSGGGSVQPGESLTLSCTGSARRFLSNYGMGWYRQFPGKMREMVAYETNRGNKRYAESVKGRFRIARDAAKTTVYLEMNSLKPEDTAVYYCNAESSGWGRRNYWGQGTQVTVSS AHP15547 CDR1 region amino acid sequence SEQ ID NO: 7 ARFLSNYG AHP15547 CDR2 region amino acid sequence SEQ ID NO: 8 ETNRGNK AHP15547 CDR3 region amino acid sequence SEQ ID NO: 9 NAESSGWGRRNY AHP15547 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 10 GCGGTGCAGCTGGTGGATAGCGGCGGCGGCAGCGTGCAGCCGGGCGAAAGCCTGACCCTGAGCTGCACCGGCAGCGCGCGTTTTCTGAGCAACTATGGCATGGGCTGGTATCGTCAGTTTCCGGGCAAAATGCGTGAAATGGTGGCGTATGAAACCAACCGTGGCAACAAACGTTAT GCGGAAAGCGTGAAAGGCCGTTTTCGTATTGCGCGTGATGCGGCGAAAACCACCGTGTATCTGGAAATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCAACGCGAAAGCAGCGGCTGGGGCCGTCGTAACTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP15580 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 11 QVQLVESGGGVVQPGGSLRLSCAASGFVLRVNTVSWYRQAPGKQRELVALITSESNEYYDDSVKGRFTISRDNAKNTIYLHMNSLKPEDTGVYFCNFITTWGQGTQVTVSS AHP15580 CDR1 region amino acid sequence SEQ ID NO: 12 GFVLRVNT AHP15580 CDR2 region amino acid sequence SEQ ID NO: 13 ITSESNE AHP15580 CDR3 region amino acid sequence SEQ ID NO: 14 NFITT AHP15580 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 15 CAGGTGCAGCTGGTGGAAAGCGGCGGCGGCGTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCGAGCGGCTTTGTGCTGCGTGTGAACACCGTGAGCTGGTATCGTCAGGCGCCGGGCAAACAGCGTGAACTGGTGGCGCTGATTACCAGCGAAAGCA ACGAATATTATGATGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCATTTATCTGCATATGAACAGCCTGAAACCGGAAGATACCGGCGTGTATTTTTGCAACTTTATTACCACCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP15662 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 16 EVQLVESGGGLVQPGGTLRLSCAASGSISRFYAVGWYRQAPGKQRELVARISSGGSTYYADSVKGRFTISINNSKNTVYLQMNNLKPEDTAVYYCNARNLWGQGTQVTVSS AHP15662 CDR1 region amino acid sequence SEQ ID NO: 17 GSISRFYA AHP15662 CDR2 region amino acid sequence SEQ ID NO: 18 ISSGGST AHP15662 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHP15662 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 20 GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCACCCTGCGTCTGAGCTGCGCGGCGAGCGGCAGCATTAGCCGTTTTTATGCGGTGGGCTGGTATCGTCAGGCGCCGGGCAAACAGCGTGAACTGGTGGCGCGTATTAGCAGCGGCGGCA GCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCATTAACAACAGCAAAAACACCGTGTATCTGCAGATGAACAACCTGAAACCGGAAGATACCGCGGTGTATTATTGCAACGCGCGTAACCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP15768 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 21 QVQLVESGGGSVQAGGSLRLSCAVSGRSISLYNMGWFRQAPGKEREFVSAINWSGGRTYYADSVKGRFTISRNNAKNTMYLQMNSLKPEDTATYYCAVDVEGWFRVSGDAAGYDYWGQGTQVTVSS AHP15768 CDR1 region amino acid sequence SEQ ID NO: 22 GRSISLYN AHP15768 CDR2 region amino acid sequence SEQ ID NO: 23 INWSGGRT AHP15768 CDR3 region amino acid sequence SEQ ID NO: 24 AVDVEGWFRVSGDAAGYDY AHP15768 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 25 CAGGTGCAGCTGGTGGAAAGCGGCGGCGGCAGCGTGCAGGCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTGAGCGGCCGTAGCATTAGCCTGTATAACATGGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTTGTGAGCGCGATTAACTGGAGCGGCGGCCGTACCTATTATGCGGATAGC GTGAAAGGCCGTTTTACCATTAGCCGTAACAACGCGAAAAACACCATGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGACCTATTATTGCGCGGTGGATGTGGAAGGCTGGTTTCGTGTGAGCGGCGATGCGGCGGGCTATGATTATTGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP15773 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 26 EVQLVESGGGLVQPGGSLRLSCLASGSDFSDSAMGWYRQAPGKQREFVAAIGAGGITTYADSVKGRFTISRDSAKNIVYLQMSSLSPEDTAVYFCNFAPGWYPPGYQYWGQGTQVTVSS AHP15773 CDR1 region amino acid sequence SEQ ID NO: 27 GSDFSDSA AHP15773 CDR2 region amino acid sequence SEQ ID NO: 28 IGAGGIT AHP15773 CDR3 region amino acid sequence SEQ ID NO: 29 NFAPGWYPPGYQY AHP15773 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 30 GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCCTGGCGAGCGGCAGCGATTTTAGCGATAGCGCGATGGGCTGGTATCGTCAGGCGCCGGGCAAACAGCGTGAATTTGTGGCGGCGATTGGCGCGGGCGGCATTACCACCTATG CGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAGCGCGAAAAACATTGTGTATCTGCAGATGAGCAGCCTGAGCCCGGAAGATACCGCGGTGTATTTTTGCAACTTTGCGCCGGGCTGGTATCCGCCGGGCTATCAGTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP15776 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 31 QVQLVESGGGLVQPGGSLTLSCAASQSISSIYLLGWYRQAPGKERESVARITTSGTTTYIADSVKGRFTLSRDNAKNTMYLQMNSLKPEDTAVYYCNVPFLVATARGNAYWGQGTQVTVSS AHP15776 CDR1 region amino acid sequence SEQ ID NO: 32 QSISSIYL AHP15776 CDR2 region amino acid sequence SEQ ID NO: 33 ITTSGTTT AHP15776 CDR3 region amino acid sequence SEQ ID NO: 34 NVPFLVATARGNAY AHP15776 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 35 CAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGACCCTGAGCTGCGCGGCGAGCCAGAGCATTAGCAGCATTTATCTGCTGGGCTGGTATCGTCAGGCGCCGGGCAAAGAACGTGAAAGCGTGGCGCGTATTACCACCAGCGGCACCACCACCTATATTG CGGATAGCGTGAAAGGCCGTTTTACCCTGAGCCGTGATAACGCGAAAAACACCATGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCAACGTGCCGTTTCTGGTGGCGACCGCGCGTGGCAACGCGTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC AHP16026 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 36 QVQLVESGGGLVQPGGSLRLSCAVLGSIEQINDINYYRWYRQPPGKQRDLVASINNSGELIYADSVKGRFFISRDKYTVYLEMRSLKAEDTAVYYCNAQDRGEYWGRGTQVTVSS AHP16026 CDR1 region amino acid sequence SEQ ID NO: 37 GSIEQIND AHP16026 CDR2 region amino acid sequence SEQ ID NO: 38 VASINNSG AHP16026 CDR3 region amino acid sequence SEQ ID NO: 39 NAQDRGEY AHP16026 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 40 CAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTGCTGGGCAGCATTGAACAGATTAACGATATTAACTATTATCGTTGGTATCGTCAGCCGCCGGGCAAACAGCGTGATCTGGTGGCGAGCATTAACAACAGCG GCGAACTGATTTATGCGGATAGCGTGAAAGGCCGTTTTTTTATTAGCCGTGATAAATATACCGTGTATCTGGAAATGCGTAGCCTGAAAGCGGAAGATACCGCGGTGTATTATTGCAACGCGCAGGATCGTGGCGAATATTGGGGCCGTGGCACCCAGGTGACCGTGAGCAGC AHP15485-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 41 EVQLVESGGGLVQPGGSLRLSCAASGRSFSNFQMGWFRQAPGKEREFVAASGWSGGPTYYADSVKGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDRVLSGRPVRYWGQGTLVTVSS AHP15485-VHH4 CDR1 region amino acid sequence SEQ ID NO: 2 GRSFSNFQ AHP15485-VHH4 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHP15485-VHH4 CDR3 region amino acid sequence SEQ ID NO: 4 YEDRVLSGRPVRY AHP15485-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 42 GAAGTGCAGCTGGTCGAGAGCGGCGGCGGCCTGGTTCAACCTGGAGGATCTCTGAGACTGTCTTGTGCTGCTTCTGGCAGAAGCTTCAGCAACTTCCAGATGGGCTGGTTTAGACAGGCCCCTGGCAAAGAGCGGGAATTCGTGGCCGCCAGCGGCTGGTCCGGCGGACCAACATACTAT GCCGACAGCGTGAAGGGCAGATTCACAATCAGCACCGACAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCTACGAGGATAGAGTGCTGAGCGGCCGCCCCGTGCGGTACTGGGGCCAGGGCACCCTCGTGACCGTGTCCAGC AHP15662-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 43 EVQLVESGGGLVQPGGSLRLSCAASGSISRFYAVGWYRQAPGKQRELVARISSGGSTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHP15662-VHH4 CDR1 region amino acid sequence SEQ ID NO: 17 GSISRFYA AHP15662-VHH4 CDR2 region amino acid sequence SEQ ID NO: 18 ISSGGST AHP15662-VHH4 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHP15662-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 44 GAAGTGCAACTGGTCGAGAGCGGCGGAGGACTGGTGCAGCCCGGCGGCTCTCTGAGACTGAGCTGCGCCGCTAGCGGCAGCATCTCTAGATTCTACGCCGTGGGCTGGTATAGACAGGCCCCTGGCAAACAGCGGGAACTGGTGGCCAGAATCAGCTCTGGCGGAA GCACCTACTACGCTGATAGCGTGAAGGGCAGATTCACCATCTCCATCGACAACAGCAAGAACACCCTCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTAATGCCCGGAACCTGTGGGGCCAGGGCACACTGGTTACAGTGTCCAGC AHP15773-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 45 EVQLVESGGGLVQPGGSLRLSCAASGSDFSDSAMGWYRQAPGKQREFVAAIGAGGITTYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYFCNFAPGWYPPGYQYWGQGTLVTVSS AHP15773-VHH4 CDR1 region amino acid sequence SEQ ID NO: 27 GSDFSDSA AHP15773-VHH4 CDR2 region amino acid sequence SEQ ID NO: 28 IGAGGIT AHP15773-VHH4 CDR3 region amino acid sequence SEQ ID NO: 29 NFAPGWYPPGYQY AHP15773-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 46 GAAGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTCCAACCTGGCGGATCTCTGAGACTGAGCTGCGCCGCTTCTGGCTCCGATTTCAGCGACAGCGCCATGGGCTGGTACAGACAGGCCCCTGGCAAACAGAGAGAGTTCGTGGCTGCTATCGGCGCCGGCGGCATCACCACCTACGCCGATAGCGTGAAGGGCAGATTCACAATCAGCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTTTTGTAATTTCGCCCCCGGCTGGTATCCTCCAGGATACCAGTACTGGGGCCAGGGCACACTCGTGACCGTGTCCAGC SEQ ID NO: 47 Amino acid sequence of ROR1 MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPY CDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPADSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRPKHVRGQNVEMSMLNAYKPKSKAKEL PLSAVRFMEELGECAFGKIYKGHLLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDDIWSFGVVLWEIFSFGLQPYYGFSNQEVI EMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPPKSRSPSSSASGSTSTGHVTSLPSGSGNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL SEQ ID NO: 48 signal peptide MLLLVTSLLLCELPHPAFLLIP SEQ ID NO: 49 Linker (used in R12 CAR as a control) (G4S)3 SEQ ID NO: 50 hinge region ESKYGPPCPPCP SEQ ID NO: 51 Transmembrane region FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 52 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 53 CD3ζ RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 54 T2A LEGGGEGRGSLLTCGDVEENPGPR SEQ ID NO: 55 tEGFR MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTK IISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 56 AHP15485 CAR MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLAQAGGSLSLSCTASGRSFSNFQMGWFRQAPGKEREFVAASGWSGGPTYYADSVKGRFTISTDDSKKTMYLQMNSLKPEDTAAYYCYEDRVLSGRPVRYWGRGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 57 AHP15580 CAR MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGVVQPGGSLRLSCAASGFVLRVNTVSWYRQAPGKQRELVALITSESNEYYDDSVKGRFTISRDNAKNTIYLHMNSLKPEDTGVYFCNFITTWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 58 AHP15662 CAR MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGTLRLSCAASGSISRFYAVGWYRQAPGKQRELVARISSGGSTYYADSVKGRFTISINNSKNTVYLQMNNLKPEDTAVYYCNARNLWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 59 AHP15773 CAR MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCLASGSDFSDSAMGWYRQAPGKQREFVAAIGAGGITTYADSVKGRFTISRDSAKNIVYLQMSSLSPEDTAVYFCNFAPGWYPPGYQYWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 60 AHP15768 CAR MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGSVQAGGSLRLSCAVSGRSISLYNMGWFRQAPGKEREFVSAINWSGGRTYYADSVKGRFTISRNNAKNTMYLQMNSLKPEDTATYYCAVDVEGWFRVSGDAAGYDYWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 61 AHP16026 CAR MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLRLSCAVLGSIEQINDINYYRWYRQPPGKQRDLVASINNSGELIYADSVKGRFFISRDKYTVYLEMRSLKAEDTAVYYCNAQDRGEYWGRGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 62 AHP15547 CAR MLLLVTSLLLCELPHPAFLLIPAVQLVDSGGGSVQPGESLTLSCTGSARFLSNYGMGWYRQFPGKMREMVAYETNRGNKRYAESVKGRFRIARDAAKTTVYLEMNSLKPEDTAVYYCNAESSGWGRRNYWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 63 AHP15776 CAR MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLTLSCAASQSISSIYLLGWYRQAPGKERESVARITTSGTTTYIADSVKGRFTLSRDNAKNTMYLQMNSLKPEDTAVYYCNVPFLVATARGNAYWGQGTQVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 64 R12 CAR MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQM NSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSD GSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEIS DGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM AHF22013 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 65 EVQLVESGGGLVQPGGSLRLSCAASGRSFSSFQMGWFRQAPGKEREFVAASGWSGGPTYYWDSVIGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDRVLSGRHVRYWGQGTLVTVSS AHF22013 CDR1 region amino acid sequence SEQ ID NO: 66 GRSFSSFQ AHF22013 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHF22013 CDR3 region amino acid sequence SEQ ID NO: 67 YEDRVLSGRHVRY AHF22016 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 68 EVQLVESGGGLVQPGGSLRLSCAASGRSFSSFQMGWFRQAPGKEREFVAASGWSGGPTYYWDSWKGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDKVLSGRRVRYWGQGTLVTVSS AHF22016 CDR1 region amino acid sequence SEQ ID NO: 66 GRSFSSFQ AHF22016 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHF22016 CDR3 region amino acid sequence SEQ ID NO: 69 YEDKVLSGRRVRY AHF22018 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 70 EVQLVESGGGLVQPGGSLRLSCAASGRSFSSFQMGWFRQAPGKEREFVAASGWSGGPTYYWDSWKGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDRVLSGRWVRYWGQGTLVTVSS AHF22018 CDR1 region amino acid sequence SEQ ID NO: 66 GRSFSSFQ AHF22018 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHF22018 CDR3 region amino acid sequence SEQ ID NO: 71 YEDRVLSGRWVRY AHP15485-VHHH4-Variant1 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 72 EVQLVESGGGLVQPGGSLRLSCAASGRSFSNFQMGWFRQAPGKEREFVAASGWSGGPTYYWDSVIGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDRVLSGRPVRYWGQGTLVTVSS AHP15485-VHHH4-Variant1 CDR1 region amino acid sequence SEQ ID NO: 2 GRSFSNFQ AHP15485-VHHH4-Variant1 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHP15485-VHHH4-Variant1 CDR3 region amino acid sequence SEQ ID NO: 4 YEDRVLSGRPVRY AHP15485-VHHH4-Variant2 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 73 EVQLVESGGGLVQPGGSLRLSCAASGRSFSSFQMGWFRQAPGKEREFVAASGWSGGPTYYWDSVIGRFTISTDNSKNTLYLQMNSLRAEDTAVYYCYEDRVLSGRPVRYWGQGTLVTVSS AHP15485-VHHH4-Variant2 CDR1 region amino acid sequence SEQ ID NO: 66 GRSFSSFQ AHP15485-VHHH4-Variant2 CDR2 region amino acid sequence SEQ ID NO: 3 SGWSGGPT AHP15485-VHHH4-Variant2 CDR3 region amino acid sequence SEQ ID NO: 4 YEDRVLSGRPVRY AHP15662-VHH4-Variant1 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 74 EVQLVESGGGLVQPGGSLRLSCAASYYISRFYAVGWYRQAPGKQRELVARISSGGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHP15662-VHH4-Variant1 CDR1 region amino acid sequence SEQ ID NO: 75 YYISRFYA AHP15662-VHH4-Variant1 CDR2 region amino acid sequence SEQ ID NO: 76 ISSGGWT AHP15662-VHH4-Variant1 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHP15662-VHH4-Variant2 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 77 EVQLVESGGGLVQPGGSLRLSCAASYFISRFYAVGWYRQAPGKQRELVARISSGGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHP15662-VHH4-Variant2 CDR1 region amino acid sequence SEQ ID NO: 78 YFISRFYA AHP15662-VHH4-Variant2 CDR2 region amino acid sequence SEQ ID NO: 76 ISSGGWT AHP15662-VHH4-Variant2 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHP15662-VHH4-Variant3 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 79 EVQLVESGGGLVQPGGSLRLSCAASHYISRFYAVGWYRQAPGKQRELVARISSGGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHP15662-VHH4-Variant3 CDR1 region amino acid sequence SEQ ID NO: 80 HYISRFYA AHP15662-VHH4-Variant3 CDR2 region amino acid sequence SEQ ID NO: 76 ISSGGWT AHP15662-VHH4-Variant3 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHF21711 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 81 EVQLVESGGGLVQPGGSLRLSCAASHSISRFYAIGWYRQAPGKQRELVARISSMGRTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHF21711 CDR1 region amino acid sequence SEQ ID NO: 82 HSISRFYA AHF21711 CDR2 region amino acid sequence SEQ ID NO: 83 ISSMGRT AHF21711 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHF21719 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 84 EVQLVESGGGLVQPGGSLRLSCAASGYISRFYAMGWYRQAPGKQRELVARISSGGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHF21719 CDR1 region amino acid sequence SEQ ID NO: 85 GYISRFYA AHF21719 CDR2 region amino acid sequence SEQ ID NO: 76 ISSGGWT AHF21719 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHF21720 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 86 EVQLVESGGGLVQPGGSLRLSCAASGYISRFYAMGWYRQAPGKQRELVARISSLGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHF21720 CDR1 region amino acid sequence SEQ ID NO: 85 GYISRFYA AHF21720 CDR2 region amino acid sequence SEQ ID NO: 87 ISSLGWT AHF21720 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL AHF21721 Single Domain Antibody Heavy Chain Variable Region Amino Acid Sequence SEQ ID NO: 88 EVQLVESGGGLVQPGGSLRLSCAASGFISRFYAMGWYRQAPGKQRELVARISSMGWTYYADSVKGRFTISIDNSKNTLYLQMNSLRAEDTAVYYCNARNLWGQGTLVTVSS AHF21721 CDR1 region amino acid sequence SEQ ID NO: 89 GFISRFYA AHF21721 CDR2 region amino acid sequence SEQ ID NO: 90 ISSMGWT AHF21721 CDR3 region amino acid sequence SEQ ID NO: 19 NARNL

Claims

1. An antibody or antigen-binding fragment thereof targeting a ROR1 protein, the antibody comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are selected from one of the following combinations: (1) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 2; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 3, The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 4, (2) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 7; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 8, The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 9, (3) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 12; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 13; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 14, (4) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 17; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 18; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (5) the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 22; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 23; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 24, (6) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 27; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 28; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 29; (7) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 32; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 33; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 34, (8) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 37; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 38; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 39; (9) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 3, The amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 67; (10) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 3, The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 71; (11) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 66; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 3, The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 4, (12) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 75; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 76; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (13) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 78; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 76; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (14) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 80; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 76; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (15) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 82; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 83; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (16) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 85; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 76; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; (17) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 85; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 87; The amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 19; and (18) The amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 89; The amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 90; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 19; Or, The antibody is a variant of the antibody defined by the amino acid sequences of HCDR1, HCDR2, and HCDR3 in any one of (1) to (18), wherein the variant comprises a total of 1 to 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid modifications in the HCDR1, HCDR2, and HCDR3 sequences compared to the antibody defined in any one of (1) to (18), or an antigen-binding fragment thereof.

2. The amino acid sequence of the heavy chain variable region is (1) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 1 or at least 90% sequence identity thereto; (2) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 6 or at least 90% sequence identity thereto; (3) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 11 or at least 90% sequence identity thereto; (4) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 16 or at least 90% sequence identity thereto; (5) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 21 or at least 90% sequence identity thereto; (6) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 26 or at least 90% sequence identity thereto; (7) A heavy chain variable region sequence having the sequence set forth in SEQ ID NO: 31 or at least 90% sequence identity thereto; (8) A heavy chain variable region sequence having the sequence set forth in SEQ ID NO: 36 or at least 90% sequence identity thereto; (9) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 65 or at least 90% sequence identity thereto; (10) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 70 or at least 90% sequence identity thereto; (11) A heavy chain variable region sequence having the sequence set forth in SEQ ID NO: 81 or at least 90% sequence identity thereto; (12) A heavy chain variable region sequence having the sequence set forth in SEQ ID NO: 84 or at least 90% sequence identity thereto; (13) A heavy chain variable region sequence having the sequence set forth in SEQ ID NO: 86 or at least 90% sequence identity thereto; and (14) The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment is selected from any one of the heavy chain variable region sequences set forth in SEQ ID NO: 88 or having at least 90% sequence identity thereto.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a single domain antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a humanized antibody.

5. The amino acid sequence of the heavy chain variable region of the humanized antibody is (1) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 41 or at least 90% sequence identity thereto; (2) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 43 or at least 90% sequence identity thereto; (3) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 45 or at least 90% sequence identity thereto; (4) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 72 or at least 90% sequence identity thereto; (5) a heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 73 or at least 90% sequence identity thereto; (6) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 74 or at least 90% sequence identity thereto; (7) A heavy chain variable region sequence having a sequence set forth in SEQ ID NO: 77 or at least 90% sequence identity thereto; and (8) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment is selected from any one of the heavy chain variable region sequences shown in SEQ ID NO: 79 or having at least 90% sequence identity thereto.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody targets the Frizzled domain or the Kringle domain of the ROR1 protein.

7. EC20 binding of the antibody to ROR1 protein, its Frizzled domain, or its Kringle domain measured by ELISA ５０ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the value is 0.1 µg / mL or less.

8. The KD value of the binding between the antibody and the ROR1 protein, its Frizzled domain, or its Kringle domain measured by surface plasmon resonance technology is 10 －６ M or less, preferably 10 －７ M or less, and more preferably 10 －８ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, having a molecular weight of M or less.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody further comprises an Fc fragment, preferably the Fc fragment is derived from human IgG, e.g., IgG1.

10. A fusion protein comprising at least one antigen-binding functional moiety, wherein the antigen-binding functional moiety comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The fusion protein of claim 10, comprising at least two antigen-binding functional moieties, each targeting the same or different antigen epitopes.

12. one of the two antigen-binding functional moieties targets the Frizzled domain of the ROR1 protein, and the other of the two antigen-binding functional moieties targets another part of the ROR1 protein other than the Frizzled domain; one of the two antigen-binding functional moieties targets the Kringle domain of ROR1, and the other of the two antigen-binding functional moieties targets another part of the ROR1 protein other than the Kringle domain; or The fusion protein of claim 10 or 11, wherein one of the two antigen-binding functional moieties targets the Kringle domain of ROR1, and the other of the two antigen-binding functional moieties targets the Frizzled domain of ROR1.

13. The fusion protein according to any one of claims 10 to 12, wherein the antigen-binding functional moieties are linked to each other via a peptide linker molecule.

14. A chimeric antigen receptor that targets the ROR1 protein, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the fusion protein according to any one of claims 10 to 13.

15. The chimeric antigen receptor of claim 14, comprising an amino acid sequence shown in any one of SEQ ID NOs: 56 to 63.

16. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the fusion protein according to any one of claims 10 to 13, or the chimeric antigen receptor according to claim 14 or 15.

17. 17. The nucleic acid molecule of claim 16, comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44 and 46.

18. 18. An expression vector comprising the nucleic acid molecule of claim 16 or 17.

19. A host cell comprising the expression vector of claim 18, or expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the fusion protein of any one of claims 10 to 13, or the chimeric antigen receptor of claim 14 or 15.

20. 20. The host cell of claim 19, which is an immune effector cell and expresses the chimeric antigen receptor of claim 14 or 15.

21. The host cell of claim 20 , wherein the immune effector cell is a T cell or an NK cell.

22. 1) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a fusion protein according to any one of claims 10 to 13, a nucleic acid molecule according to claim 16 or 17, or a host cell according to any one of claims 19 to 21; 2) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

23. A method for treating a disease, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the fusion protein of any one of claims 10 to 13, the nucleic acid molecule of claim 16 or 17, the host cell of any one of claims 19 to 21, or the pharmaceutical composition of claim 22.

24. 24. The method of claim 23, wherein the disease is a tumor that expresses the ROR1 protein.

25. 25. The method of claim 24, wherein the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma, and ovarian cancer.

26. A kit for detecting ROR1 protein in a sample, the kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a fusion protein according to any one of claims 10 to 13.

27. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the fusion protein according to any one of claims 10 to 13, the nucleic acid molecule according to claim 16 or 17, or the host cell according to any one of claims 19 to 21 in the manufacture of a medicament for treating a tumor.

28. The use according to claim 27, wherein the disease is a tumor that expresses the ROR1 protein.

29. 29. The use according to claim 28, wherein the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma and ovarian cancer.

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