Single-domain antibody targeting human ROR1

Abstract

Provided herein are single domain antibodies targeting ROR1 protein, as well as chimeric antigen receptors (CARs) and CAR-T cells comprising the single domain antibodies. The invention further provides application of the single-domain antibody and the CAR-T cell in treatment of tumors expressing ROR1 protein.

Description

Single-domain antibody targeting human ROR1

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202211370878.5, filed on November 3, 2022, and Chinese patent application number 202310575171.6, filed on May 18, 2023, and the above applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of tumor immunotherapy and molecular immunology, and specifically relates to the development of a high-affinity, high-specificity, multi-antigen recognition epitope-targeted human ROR1 CAR-T with higher functionality. Background Art

[0004] Tumor immunotherapy has become one of the most important means of tumor treatment. Only after identifying cancer cells can the immune system effectively attack cancer cells. Canceration is the result of normal somatic cells losing their normal cell regulatory functions and gene mutations accumulating to a certain degree. However, cancer cells can disguise themselves as normal somatic cells and cleverly avoid the attack of the immune system.

[0005] Tumor-associated antigens (TAAs) are self-antigens expressed by tumor cells. They are partially present in normal host cells, primarily through gene amplification or post-translational modification, but tend to be highly or characteristically expressed in tumor cells. Currently, many tumor-associated and tumor-specific antigens have been discovered, and targeted therapy targeting TAAs is an important approach to cancer treatment. Several cancer immunotherapy drugs that rely on this mechanism are already on the market and are very effective clinically.

[0006] Monoclonal antibodies (mAbs) have become an increasingly important class of drugs, and their clinical application has revolutionized the field of cancer treatment. Different mAbs have different anti-tumor mechanisms of action, including blocking tumor-specific growth factor receptors or immunomodulatory molecules, as well as complement- and cell-mediated tumor cell lysis. Rituximab targets the CD20 antigen on B cells for the treatment of non-Hodgkin lymphoma, and trastuzumab (Herceptin) targets HER2 for the treatment of HER2-positive breast cancer are just two well-known examples. The development of drugs targeting TAAs remains an active area of ​​research. Currently, a large number of targeted drugs are undergoing preclinical or clinical testing for a variety of tumor types.

[0007] Generally speaking, antibody-mediated TAA recognition can cleverly guide NK cells or T cells to target tumor cells with high TAA expression. Antibodies targeting TAAs (e.g., rituximab and trastuzumab) can not only directly kill tumor cells through ADCC, but can also serve as diagnostic markers or innovatively increase the targeting of traditional cancer therapies.

[0008] Adoptive immunotherapy is a transformative treatment approach in hematology. It involves genetically engineering T cells to express synthetic chimeric antigen receptors (CARs). CARs are engineered receptors that redirect immune cells to target cancer cells. CAR-T cells have achieved impressive results in patients with hematological malignancies, with six FDA-approved CAR-T cells for the treatment of 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 regions, costimulatory domains, and activation domains) are systematically engineered to optimize cell activation and enhance persistence. Using different domains or making minor modifications to the domain sequences can significantly alter the efficacy of CAR-T cells. Despite the success of CAR-T in the treatment of hematological malignancies to date, many patients experience CAR-T cell-related toxicities and relapse from CAR-T cell therapy, which may be related to the CAR binding epitope, highlighting the need to develop antibodies that bind to multiple epitopes. The new generation of CAR-T cells is designed to reduce toxicity and prevent relapse by refining antigen targeting, regulating the assembly or activation of CAR components, preventing anti-CAR immunity, and / or protecting cells from responding to their surrounding environment. Although FDA-approved CAR-T cells target two B cell antigens, CD19 and B cell maturation antigen (BCMA), other targets for B cell malignancies, other blood cancers, and solid tumors are rapidly emerging. Especially for solid tumors, there is an urgent need to develop new CART therapies to meet the unmet clinical treatment needs of patients.

[0009] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that, as an orphan receptor tyrosine kinase-like surface antigen, is expressed by many tissues during embryogenesis and plays a role in embryonic skeletal, cardiopulmonary, and neural development. ROR1 is primarily expressed in embryonic tissues; its expression levels in adult tissues are restricted, including the parathyroid glands, pancreatic islets, and the esophageal, gastric, and duodenal regions. 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 minimal residual disease in CLL. Higher ROR1 expression levels are associated with a higher number of circulating leukemic cells and disease aggressiveness, and ROR1 expression is retained on 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 leukaemia,British Journal of Haematology,Volume190,Issue6,September 2020,Pages e346-e349).

[0010] Summary of the Invention

[0011] In one aspect, provided herein is an antibody or antigen-binding fragment thereof targeting 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:

[0012] (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2;

[0013] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 3;

[0014] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 4;

[0015] (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 7;

[0016] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 8;

[0017] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 9;

[0018] (3) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 12;

[0019] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 13;

[0020] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 14;

[0021] (4) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 17;

[0022] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 18;

[0023] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0024] (5) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 22;

[0025] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 23;

[0026] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 24;

[0027] (6) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 27;

[0028] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 28;

[0029] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 29;

[0030] (7) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 32;

[0031] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 33;

[0032] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 34; and

[0033] (8) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 37;

[0034] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 38;

[0035] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 39;

[0036] (9) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 66;

[0037] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 3;

[0038] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 67;

[0039] (10) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 66;

[0040] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 3;

[0041] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 71;

[0042] (11) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 66;

[0043] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 3;

[0044] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 4;

[0045] (12) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 75;

[0046] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 76;

[0047] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0048] (13) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 78;

[0049] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 76;

[0050] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0051] (14) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 80;

[0052] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 76;

[0053] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0054] (15) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 82;

[0055] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 83;

[0056] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0057] (16) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 85;

[0058] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 76;

[0059] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0060] (17) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 85;

[0061] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 87;

[0062] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0063] (18) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 89;

[0064] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 90;

[0065] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19;

[0066] or,

[0067] 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 at least 1 and no more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in the HCDR1, HCDR2 and HCDR3 sequences compared to the antibody defined in any one of (1) to (18).

[0068] In some embodiments, the amino acid sequence of the heavy chain variable region is selected from any one of the following:

[0069] (1) a heavy chain variable region sequence represented by SEQ ID NO: 1 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0070] (2) a heavy chain variable region sequence represented by SEQ ID NO: 6 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0071] (3) a heavy chain variable region sequence represented by SEQ ID NO: 11 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0072] (4) a heavy chain variable region sequence represented by SEQ ID NO: 16 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0073] (5) a heavy chain variable region sequence represented by SEQ ID NO: 21 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0074] (6) the sequence of SEQ ID NO: 26 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0075] (7) a heavy chain variable region sequence represented by SEQ ID NO: 31 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0076] (8) the sequence of SEQ ID NO: 36 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0077] (9) the sequence of SEQ ID NO: 65 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0078] (10) the sequence of SEQ ID NO: 68 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0079] (11) the sequence of SEQ ID NO: 70 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0080] (12) the sequence of SEQ ID NO: 81 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0081] (13) the sequence of SEQ ID NO: 84 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0082] (14) a heavy chain variable region sequence represented by SEQ ID NO: 86 or a heavy chain variable region sequence having at least 90% sequence identity thereto; and

[0083] (15) The sequence shown in SEQ ID NO: 88 or a heavy chain variable region sequence having at least 90% sequence identity thereto.

[0084] In some embodiments, the antibody is a single domain antibody.

[0085] In some embodiments, the antibody is a humanized antibody.

[0086] In some embodiments, the amino acid sequence of the heavy chain variable region of the humanized antibody is selected from any one of the following:

[0087] (1) a heavy chain variable region sequence represented by SEQ ID NO: 41 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0088] (2) a heavy chain variable region sequence represented by SEQ ID NO: 43 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0089] (3) The sequence of SEQ ID NO: 45 or a heavy chain variable region sequence having at least 90% sequence identity thereto

[0090] (4) a heavy chain variable region sequence represented by SEQ ID NO: 72 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0091] (5) a heavy chain variable region sequence represented by SEQ ID NO: 73 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0092] (6) a heavy chain variable region sequence represented by SEQ ID NO: 74 or a heavy chain variable region sequence having at least 90% sequence identity thereto;

[0093] (7) a heavy chain variable region sequence represented by SEQ ID NO: 77 or a heavy chain variable region sequence having at least 90% sequence identity thereto; and

[0094] (8) The sequence shown in SEQ ID NO: 79 or a heavy chain variable region sequence having at least 90% sequence identity thereto.

[0095] In some embodiments, the antibody targets the Frizzled domain or the Kringle domain of the ROR1 protein.

[0096] In some embodiments, the EC binding of the antibody to ROR1 protein, its Frizzled domain or its Kringle domain as determined by ELISA is 50 The value is no higher than 0.1μg / mL.

[0097] In some embodiments, the antibody binds to the ROR1 protein, its Frizzled domain, or its Kringle domain with a KD value of no greater than 10 as determined by surface plasmon resonance. -6 M, preferably not higher than 10 -7 M, more preferably not higher than 10 -8 M.

[0098] In some embodiments, the antibody further comprises an Fc fragment; preferably, the Fc fragment is derived from human IgG, such as IgG1.

[0099] In another aspect, provided herein is a fusion protein comprising at least one antigen-binding functional portion, wherein the antigen-binding functional portion comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0100] In some embodiments, the fusion protein includes at least two antigen-binding functional parts, and the two antigen-binding functional parts target the same or different antigen epitopes, respectively.

[0101] In some embodiments, one of the two antigen-binding functional portions targets the Frizzled domain of the ROR1 protein, and the other of the two antigen-binding functional portions targets other parts of the ROR1 protein except the Frizzled domain; one of the two antigen-binding functional portions targets the Kringle domain of ROR1, and the other of the two antigen-binding functional portions targets other parts of the ROR1 protein except the Kringle domain; or one of the two antigen-binding functional portions targets the Kringle domain of ROR1, and the other of the two antigen-binding functional portions targets the Frizzled domain of ROR1.

[0102] In some embodiments, the antigen-binding functional portions are connected via a peptide linker molecule.

[0103] In another aspect, provided herein is a chimeric antigen receptor targeting ROR1 protein, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain is the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned fusion protein.

[0104] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 56-63.

[0105] In another aspect, provided herein is a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, fusion protein, or chimeric antigen receptor as claimed above.

[0106] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44, and 46.

[0107] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule of claim 1 .

[0108] In another aspect, provided herein is a host cell comprising the above-mentioned expression vector or expressing the above-mentioned antibody or antigen-binding fragment thereof, fusion protein or chimeric antigen receptor.

[0109] In some embodiments, the host cell is an immune effector cell and expresses the chimeric antigen receptor as claimed in claim 1.

[0110] In some embodiments, the immune effector cells are T cells or NK cells.

[0111] In another aspect, provided herein is 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.

[0112] In another aspect, provided herein is a method for treating a disease, comprising administering an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, host cell or pharmaceutical composition to a subject in need thereof.

[0113] In some embodiments, the disease is a tumor expressing ROR1 protein.

[0114] In some embodiments, the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma, and ovarian cancer.

[0115] In another aspect, the present invention provides a kit for detecting ROR1 protein in a sample, wherein the kit comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned fusion protein.

[0116] In another aspect, the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule or host cell in the preparation of a medicament for treating tumors.

[0117] In some embodiments, the disease is a tumor expressing ROR1 protein.

[0118] In some embodiments, the tumor is selected from chronic lymphocytic leukemia, mantle cell lymphoma, and ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1: ELISA results of purified monoclonal antibody and human ROR1-His recombinant protein.

[0120] Figure 2: ELISA results of purified monoclonal antibody and human ROR1 Kringle-His recombinant protein.

[0121] Figure 3: ELISA results of purified monoclonal antibody and human ROR1-His recombinant protein.

[0122] Figure 4: FACS analysis of purified monoclonal antibodies binding to cell lines expressing human and mouse ROR1.

[0123] Figure 5: Affinity test results of humanized antibodies and their corresponding human ROR1-related proteins.

[0124] Figure 6: FACS results of humanized antibody binding to cell lines expressing human and mouse ROR1.

[0125] Figure 7: Flow cytometry results of the human ROR1 engineered cell line CHO-K1 / ROR1.

[0126] Figure 8: Schematic diagram of the CAR structure constructed in the present invention.

[0127] Figure 9: Flow cytometry analysis of T cell proportions after PBMC activation for 4 days.

[0128] Figure 10: Evaluation of the activation effect of human ROR1-CAR targeting on Jurkat cells based on the reporter gene method.

[0129] Figure 11: Evaluation of the activation effect of human ROR1-CAR on Jurkat cells based on the secretion level of cytokine IL-2.

[0130] Figure 12: Specific killing effect of human ROR1-CAR-T targeting target cells CHO-K1 / ROR1 / Luc.

[0131] Figure 13: Detection of cytokine release levels after co-incubation of human ROR1-targeted CAR-T with target cells CHO-K1 / ROR1 / Luc.

[0132] Figure 14: Affinity test results of the AHP15485-VHH4 series affinity matured antibodies and their corresponding human ROR1-related proteins.

[0133] Figure 15: Affinity test results of the AHP15662-VHH4 series affinity matured antibodies and their corresponding human ROR1-related proteins. DETAILED DESCRIPTION

[0134] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0135] The term "or" refers to a single element of the listed 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.

[0136] The term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0137] The terms "comprising" or "including" refer to including the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. As used herein, when the terms "comprising" or "including" are used, unless otherwise indicated, they also encompass situations consisting of the stated elements, integers, or steps. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0138] ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved membrane protein that is 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 membrane-proximal Kringle domain; a transmembrane domain; and 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 or mouse ROR1 proteins.

[0139] As used herein, the term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and is a protein or polypeptide that exhibits binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in the amino acid sequences, the heavy chain and light chain are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cellular cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions, each with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody-antigen binding and are therefore also referred to as complementary determining regions (CDRs). The amino acid sequences of the CDRs can be easily determined using numbering schemes recognized in the art, such as Kabat, Chothia, IMGT, AbM or Contact. The three complementary determining regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3, and the three complementary determining regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3. Each heavy chain variable region (VH) and light chain variable region (VL) can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In a specific embodiment, the CDR sequences of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0140] An "antigen-binding fragment" of an antibody molecule refers to a polypeptide that includes a partial sequence (particularly the CDR sequence) of a source antibody and possesses the binding specificity of the source antibody. This antigen-binding fragment typically includes at least the heavy chain variable region of the source antibody and possesses antigen-binding ability. Antigen-binding fragments come in various forms, such as Fab, Fab', F(ab')2, single-chain antibodies (scFv), and single-domain antibodies (sdAb). Those skilled in the art know how to obtain these antigen-binding fragments. For example, a classic antibody molecule can be digested with papain to produce a Fab fragment, and with pepsin to produce F(ab')2. Treatment with a reducing agent breaks the disulfide bonds between the hinge regions of the F(ab')2 to form a Fab' fragment. A "single-chain antibody (scFv)" is composed of the variable regions of the heavy and light chains of an antibody, linked by a short peptide, into a single peptide chain. Proper folding allows the variable regions from the heavy and light chains to interact through non-covalent bonds to form the Fv fragment, allowing the scFv to retain its affinity for the antigen.

[0141] "Single domain antibody (sdAb)", or also known as "V H "H antibody" refers to an antibody molecule with antigen binding ability, including heavy chain variable region but no light chain. Structurally, single-domain antibodies can also be considered as fragments of classic four-chain antibody molecules. Single-domain antibodies were first discovered in camelids. Subsequently, researchers discovered more single-domain antibodies with antigen binding ability through screening of antibody libraries (such as phage display libraries). Single-domain antibodies have some advantages over ordinary antibody molecules (such as classic antibody molecules), including but not limited to: smaller molecular weight, easy to reach tissues or parts that ordinary antibody molecules cannot reach when used in the human body, or able to access antigen epitopes in proteins or polypeptides that ordinary antibody molecules cannot reach; more stable, able to withstand changes in temperature and pH, as well as the effects of denaturants and proteases.

[0142] The term "fusion protein" refers to a protein molecule composed of at least two different peptide segments that is artificially generated (e.g., by genetic engineering technology). These peptide segments do not exist in nature, or do not exist in the same protein molecule. Common examples of fusion proteins including antibody fragments include multispecific antibodies, antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also known as immunotoxins), enzyme-labeled antibodies for immunoassays, chimeric antigen receptors (CARs), and the like. In one embodiment, the fusion protein is a multispecific antibody comprising at least two single domain antibodies provided herein, which can bind to different antigenic epitopes on the ROR1 protein.

[0143] An "epitope," also known as an "antigenic determinant," refers to the site on an antigen that binds to a corresponding antibody molecule. Epitopes can be either sequence or conformational. Sequence epitopes consist of consecutively arranged amino acid residues. Conformational epitopes consist of discontinuously arranged amino acid residues that are spatially close to each other to form a specific conformation. For example, in a polypeptide, 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.

[0144] "Fc fragment" refers to the handle region of the "Y"-shaped antibody molecule, that is, the crystallizable fragment (Fc) includes the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing the antibody molecule by proteolytic enzymes (such as papain). In some examples, the Fc region may include a hinge, CH2 and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment can be from IgG, IgM, IgD, IgE or IgA. In some examples, the Fc region is from IgG1, IgG2, IgG3 or IgG4. "Fc fragment" also includes variant Fc fragments from natural Fc fragments that have been modified but still retain their effector functions. "Variant Fc fragment" comprises at least one amino acid sequence in the natural Fc fragment. Amino acid sequence of amino acid changes. In some instances, 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 acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region 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 functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediated phagocytosis, etc.

[0145] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using the default settings. In some embodiments, "at least 90% sequence identity" as described herein includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0146] For antibodies or antigen-binding fragments thereof, "targeting", "directed against" or "specific 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) relative to other molecules present in the environment. A molecule can target, direct against or specifically bind to more than one molecule, for example, a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding affinity of an antibody to an antigen can be measured by a number of parameters, such as the EC of the antibody to the antigen. 50 value or KD value.

[0147] EC 50 Concentration for 50% of maximal effect refers to the concentration that causes 50% of the maximum effect. In enzyme-linked immunosorbent assays (ELISAs), when used to indicate the binding ability of an antibody molecule to its corresponding antigen, it refers to the concentration of the antibody molecule that produces half the maximum detection signal (such as colorimetric or fluorescence intensity). EC 50 The lower the value, the greater the binding affinity to the antigen.

[0148] KD value can also be used to measure the binding affinity between an antibody and its antigen. KD value is the equilibrium dissociation constant between an antibody and its antigen, i.e., k off / k onTherefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.

[0149] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Full-length proteins and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (which are generally conservative in practice), as long as the protein retains the desired activity. These modifications may be purposeful, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0150] When referring to an antibody or its antigen-binding fragment, the term "variant" as used herein refers to a protein obtained by introducing one or more amino acid insertions, deletions or substitutions into a parent antibody molecule, which still retains at least part of the function of the parent antibody molecule (especially the function 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%, 90% of the antigen binding ability of its parent antibody molecule, or 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 even 100% or more of the antigen binding affinity of its parent antibody molecule. For an antibody molecule or its antigen-binding fragment, a variant generally includes amino acid changes in the variable region framework sequence and / or constant region, but does not exclude that one or a few amino acid changes may be made to the CDR region sequence. Therefore, those skilled in the art will also understand that, based on the specific antibody sequences provided herein, a small number of amino acids can be replaced, deleted, added, and the binding ability or biological activity of the resulting products to the corresponding antigen (ROR1 protein) can be verified or screened to obtain corresponding variants of the anti-ROR1 protein antibody molecules provided by the present invention. These variants should also be included within the scope of the present invention.

[0151] "Chimeric antigen receptor (CAR)" refers to an engineered membrane protein receptor molecule herein that can confer desired specificity to immune effector cells, such as the ability to bind to specific tumor antigens. Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen binding domain is a scFv sequence or a single domain antibody fragment that is responsible for recognizing and binding to a specific antigen. The intracellular signaling domain generally includes an immunoreceptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3ζ molecule, which is responsible for activating immune effector cells and producing a killing effect. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus that is responsible for the localization of the nascent protein in the cell, and a hinge region between the antigen binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also include a costimulatory domain derived from, for example, a 4-1BB or CD28 molecule.

[0152] "CAR cells" herein refer to cells that express CAR molecules on their surface. In most cases, the cells are immune cells, such as T cells or NK cells. Accordingly, T cells expressing CAR are referred to herein as "CAR-T" or "CAR-T cells." In addition, when referring to CAR-T cells herein, unless otherwise specified, they refer not only to cells that are directly modified with CAR, but also to daughter cells produced after these cells proliferate in vitro or in vivo.

[0153] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such 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 a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0154] The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus). A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0155] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as 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 their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. In some embodiments, the single-domain antibodies provided herein can be produced by secretion from mammalian cells. Host cells include the offspring of a single host cell, and the offspring may not necessarily be completely identical to the original parent cell (in terms of morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Cells or cell lines from which host cells can be isolated also include cells transfected with nucleic acid molecules or expression vectors provided herein in vivo. In one embodiment, the host cell is a CAR cell, such as a CAR-T cell.

[0156] "Subject" includes animals, such as mammals, including but not limited to primates, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian experimental animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The subject can be male or female and can be any age-appropriate subject, including infants, young children, young people, adults, and elderly subjects. In some instances, the subject refers to an individual who needs to diagnose or treat a disease or condition. In some instances, the subject receiving diagnosis or treatment can be a patient who suffers from a condition associated with the diagnosis or treatment, or is at risk of developing the condition. In a specific instance, the subject is a human, such as a human patient. The term is generally used interchangeably with "patient," "test subject," "treatment subject," etc.

[0157] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein.

[0158] When referring to disease treatment, "effective amount" refers to the amount of active compound (such as antibody) sufficient to cause the biological or medical response desired by the clinician in the subject. The "effective amount" when administering the antibody provided herein can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight. The methods of administration of the active compounds (such as antibodies, fusion proteins) or immune effector cells herein include, but are not limited to, injection, for example, by intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, etc.

[0159] Antibodies or antigen-binding fragments thereof targeting ROR1 protein

[0160] Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ROR1 protein. The antibody or antigen-binding fragment thereof binds to ROR1 protein (or its Frizzled domain or Kringle domain) with a relatively high binding affinity. For example, as described in the Examples below, the binding ability of the antibody or antigen-binding fragment thereof to ROR1 protein (or its Frizzled domain or Kringle domain) can be measured by assays such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In addition, it can also be measured by other protein interaction assays known in the art, for example, biofilm layer interferometry (BLI) technology.

[0161] In some embodiments, the antibody is a single-domain antibody. In some embodiments, the single-domain antibody is obtained by screening a natural alpaca library (single-domain antibody phage display library) with ROR1 protein (or its Frizzled domain or Kringle domain). In other embodiments, after the antibody sequence is known, the single-domain antibody is obtained by genetic engineering technology, for example, by introducing an expression vector expressing the antibody or its antigen-binding fragment into a host cell and culturing the host cell.

[0162] Provided herein are heavy chain CDR sequences of antibodies targeting ROR1 protein (or its Frizzled domain or Kringle domain), which are shown in SEQ ID NO: 2-4, 7-9, 12-14, 17-19, 22-24, 27-29, 32-34 or 37-39, respectively.

[0163] Based on the CDR sequences provided herein, those skilled in the art can construct various polypeptide constructs (including antibodies or antigen-binding fragments thereof) that have the ability to bind to ROR1 protein (or its frizzled domain or Kringle domain), which includes using framework regions (FR) and / or constant regions from different antibody molecules in combination with these CDR sequences. These framework regions include natural framework region sequences from human antibodies or animal (such as mouse, rat, sheep, camel, etc.) antibodies. These framework regions can also include framework region sequence variants generated by altering the natural framework region sequence. By combining the CDR sequences provided herein with different framework region sequences to form heavy chain variable regions and testing their binding ability to ROR1 protein (or its frizzled domain or Kringle domain), polypeptide constructs that specifically bind to ROR1 protein (or its frizzled domain or Kringle domain) can be easily obtained.

[0164] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof targeted to ROR1 protein (or its Frizzled domain or Kringle domain) provided herein 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 shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31 or 36.

[0165] In some embodiments, humanized antibodies of the above-mentioned single-domain antibodies are also provided herein. These humanized antibodies are substantially identical to the above-mentioned single-domain antibodies in CDR sequence, but the alpaca antibody framework region is replaced by the human antibody framework region.

[0166] In some embodiments, the heavy chain variable region of a humanized antibody or antigen-binding fragment thereof targeted to a ROR1 protein (or its frizzled domain or Kringle domain) provided herein 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 shown in SEQ ID NO: 41, 43 or 45.

[0167] Based on the above-mentioned single-domain antibodies (including humanized single-domain antibodies), we also performed affinity maturation mutations and screened some clones with improved affinity.Accordingly, in some embodiments, we provide affinity matured antibodies whose CDR sequences are as follows: (1) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 66, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 67; (2) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 66, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 69; (3) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 66, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 71; (4) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 66, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 4; (5) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 75, the amino acid sequence of HCDR2 is shown in SEQ ID NO: NO: 76, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (6) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 78, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 76, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (7) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 80, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 76, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (8) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 82, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 83, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (9) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 85, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 76, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (10) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 85, the amino acid sequence of HCDR2 is shown in SEQ ID NO: NO: 87, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; (11) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 89, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 90, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19.

[0168] In some embodiments, the heavy chain variable region of the affinity matured antibodies targeted to ROR1 protein (or its frizzled domain or Kringle domain) provided herein 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 shown in SEQ ID NO: 65, 68, 70, 72, 73, 74, 77, 79, 81, 84, 86 or 88.

[0169] It will be understood by those skilled in the art that, based on the specific sequences provided herein, a small number of amino acids can be replaced, deleted, added, and the resulting products can be verified or screened for their binding ability or biological activity to the ROR1 protein (or its Frizzled domain or Kringle domain), thereby obtaining corresponding variants of the antibody molecules targeting the ROR1 protein (or its frizzled domain or Kringle domain) provided herein, and these variants should also be included within the scope of the present invention. For example, the antibody molecules provided herein may have at least one and no more than ten, for example, no more than 5, 4, 3, 2, or 1 amino acid changes in their full-length or variable region sequence or CDR sequence. For example, there can be at least one and no more than 10, such as no more than 5, 4, 3, 2 or 1 amino acid changes in the heavy chain variable region sequence shown in SEQ ID NO: 1, 6, 11, 16, 21, 26, 31 or 36, and there can also be no more than 5, 4, 3, 2 or 1 amino acid changes in the CDR sequences 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, or the antibody has any combination of the above modifications.

[0170] It is expected that the antibodies or antigen-binding fragments thereof described herein may comprise conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as substitutions of one amino acid residue by another amino acid residue of similar chemical structure, with little or substantially no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions can, for example, be substitutions of one amino acid from the following groups (a)-(e) by another amino acid from the same group: (a) small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0171] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination-like, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, the modified soluble polypeptide may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which involves the attachment of one or more sialic acid groups to a polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins while also reducing the potential immunogenicity of proteins.

[0172] Fusion protein

[0173] Provided herein are fusion proteins containing at least one antibody or antigen-binding fragment thereof that specifically binds to a ROR1 protein (or its Frizzled domain or Kringle domain) provided herein and at least one other functional portion. Examples of such fusion proteins include, but are not limited to, multispecific antibodies (such as bispecific antibodies) and chimeric antigen receptors (CARs).

[0174] 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 and 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 formed by the antibody or antigen-binding fragment thereof and the Fc fragment has the ability to specifically bind to the ROR1 protein (or its Frizzled domain or Kringle domain) and at the same time has the effector function of the Fc fragment, such as mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, etc. In addition, fusion with the Fc fragment can increase the half-life of the antibody or antigen-binding fragment thereof in vivo, so as to increase the dosing interval when the antibody or antigen-binding fragment thereof is used as a therapeutic drug.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof can be linked to a protein tag to form a fusion protein. Protein tags may include purification tags and detectable tags. Purification tags include, but are not limited to, His6 tags, Flag tags, MBP tags, GST tags, SUMO tags, and the like. Detectable tags 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 information of ROR1 protein (or its Frizzled domain or Kringle domain) in a subject's body or within a cell. Examples of detectable tags include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), and the like; and fluorescent proteins, such as GFP. Due to the specific binding ability of the antibody or antigen-binding fragment thereof to the ROR1 protein (or its Frizzled domain or Kringle domain), the amount of the antibody or antigen-binding fragment thereof can be determined by the amount of the detectable tag linked to the antibody or antigen-binding fragment thereof, and thereby the content of the ROR1 protein (or its Frizzled domain or Kringle domain) in the sample.

[0176] 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 this case, the specific binding ability of the antibody or antigen-binding fragment thereof to the ROR1 protein (or its Frizzled domain or Kringle domain) can be used to purposefully deliver the cytokine or therapeutic protein to specific tissues or cells (e.g., tumor tissue expressing the ROR1 protein (or its Frizzled domain or Kringle domain)), thereby achieving the therapeutic effect of the cytokine or therapeutic protein.

[0177] In some embodiments, the fusion protein is a bispecific antibody, wherein one antigen-binding portion targets the ROR1 protein (or its Frizzled domain or Kringle domain) (e.g., from a single domain antibody provided herein), and the other antigen-binding portion can bind to a second antigen or protein other than the ROR1 protein (or its Frizzled domain or Kringle domain). In other embodiments, the fusion protein is a bispecific antibody, wherein one antigen-binding portion targets the ROR1 protein (or its Frizzled domain or Kringle domain), and the other antigen-binding portion also targets the ROR1 protein (or its Frizzled domain or Kringle domain) (both from a single domain antibody provided herein), but the two antigen-binding portions differ in amino acid sequence (especially CDR sequence) and respectively bind to different antigenic epitopes on the ROR1 protein (or its Frizzled domain or Kringle domain). In one embodiment, one antigen-binding portion targets the Frizzled domain, and another antigen-binding portion targets a site on the ROR1 protein other than the Frizzled domain; in another embodiment, one antigen-binding portion targets the Kringle domain, and another antigen-binding portion targets a site on the ROR1 protein other than the Kringle domain; in another embodiment, one antigen-binding portion targets the Kringle domain, and another antigen-binding portion targets the Frizzled domain.

[0178] In some embodiments, the two antigen-binding moieties in the bispecific antibody are connected in series via a peptide linker (or linker sequence). The peptide linker may be a naturally occurring linker, a synthetic linker, or a combination of the two. Particularly suitable linker sequences primarily include amino acid residues 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) (such as 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 only Gly, Ser, Ala, and / or Thr residues. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, the peptide linker comprises only glycine residues. In some examples, the peptide linker comprises only glycine and serine residues, such as (GS) nThe fusion protein formed by connecting 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 chimeric antigen receptor with bispecificity.

[0179] 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 inhibiting a signaling pathway in an immune cell. In some embodiments, the second antigen is a T cell surface molecule, such as a component of a T cell receptor complex, for example CD3 (including γ, δ, ε, ζ, and η chains).

[0180] Chimeric Antigen Receptor (CAR)

[0181] Herein, the CAR may comprise an extracellular antigen binding domain that specifically binds to the ROR1 protein (or its Frizzled domain or Kringle domain), a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

[0182] In some embodiments, the extracellular antigen binding domain of the CAR may include a single domain antibody (or its antigen binding fragment) provided herein. The single domain antibody (or its antigen binding fragment) may be connected to the transmembrane domain through a hinge region, such as a CD8α hinge. The CAR can be used to transduce immune effector cells (e.g., T cells) and express them on the cell surface. Thus, the present invention also provides T cells expressing the chimeric antigen receptor, and the use of the T cells and / or the CAR for the preparation of a drug for treating related ROR1 protein diseases.

[0183] In some embodiments, the extracellular antigen-binding domain may comprise two or more single-domain antibodies (or antigen-binding fragments thereof) targeting ROR1 protein (or its Frizzled domain or Kringle domain) provided herein. These single-domain antibodies (or antigen-binding fragments thereof) are connected in series directly or via a peptide linker. Preferably, these single-domain antibodies (or antigen-binding fragments thereof) target different antigenic epitopes on the ROR1 protein, respectively. 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 other sites on the ROR1 protein except the Frizzled domain; one single-domain antibody (or antigen-binding fragment thereof) targets the Kringle domain, and another single-domain antibody (or antigen-binding fragment thereof) targets other sites on the ROR1 protein except the Kringle domain; 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.

[0184] CAR provided herein may include a transmembrane domain, which may include a polypeptide selected from the group consisting of: α, β or ζ chain of a T cell receptor, CD28, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In a specific example, the transmembrane domain may include an amino acid sequence shown in SEQ ID NO: 51 or a functional variant thereof.

[0185] The CAR provided herein may include a costimulatory domain, which may include a polypeptide selected from the group consisting of CD28, 4-1BB, OX40, and ICOS. In a specific example, the costimulatory domain may include an amino acid sequence shown in SEQ ID NO: 52 or a functional variant thereof.

[0186] The CAR provided herein may include an intracellular signaling domain, which may include a signaling domain from CD3 zeta. In a specific example, the intracellular signaling domain may include an amino acid sequence shown in SEQ ID NO: 53 or a functional variant thereof.

[0187] The CAR provided herein may include a hinge region that connects the extracellular antigen binding domain of the ROR1 protein (or its Frizzled domain or Kringle domain) and the transmembrane domain. In a specific example, the hinge region may include the amino acid sequence shown in SEQ ID NO: 50 or a functional variant thereof.

[0188] The CAR provided herein may include a signal peptide, which may, for example, be located at the N-terminus of an extracellular antigen binding domain that specifically binds to a ROR1 protein (or its Frizzled domain or Kringle domain). The signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 48 or a functional variant thereof.

[0189] The CAR provided herein can also be connected to a cleavage peptide. In one embodiment, the cleavage peptide may comprise an amino acid sequence from a T2A peptide. In a specific example, the cleavage peptide may comprise an amino acid sequence shown in SEQ ID NO: 54 or a functional variant thereof.

[0190] In some embodiments, the CAR can also be connected to a tEGFR fragment by a cleavage peptide, and the tEGFRt fragment can be used for signal detection (e.g., indicating a CAR positive cell), or used as a molecular switch for a CAR-T cell. In a specific example, the tEGFR fragment comprises the amino acid sequence shown in SEQ ID NO:55 or a functional variant thereof.

[0191] In some specific embodiments, the CAR includes the amino acid sequence shown in any one of SEQ ID NOs: 56-63. It should be understood that these listed sequences also include signal peptide sequences and tEGFR fragments connected by cleavage peptides, and these parts are not functional parts of the CAR and do not exist in the form of a fusion protein with the remaining parts after expression in T cells. Therefore, in other specific embodiments, the CAR provided herein includes the remaining sequence after removing the signal peptide and / or the tEGFR fragment connected by the cleavage peptide from the amino acid sequence shown in any one of SEQ ID NOs: 56-63.

[0192] Nucleic acids, vectors, cells and pharmaceutical compositions

[0193] Provided herein are isolated nucleic acid molecules that can encode the antibodies or antigen-binding fragments thereof, fusion proteins, or CARs described above. In some embodiments, the isolated nucleic acid molecule may comprise a nucleic acid sequence or a functional variant thereof shown in any one of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 42, 44, and 46. These nucleic acid molecules can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cleavage and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0194] Also provided herein is a vector that may contain the above-mentioned 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 the CAR is placed on a lentiviral expression vector for the preparation of CAR-T cells. For example, the lentiviral vector may contain a nucleic acid sequence shown 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 different nucleotide sequence encoding the same amino acid sequence due to codon degeneracy. In addition, the vector may also contain other genes, such as a marker gene that allows the vector to be selected in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed 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. On the other hand, the present application provides an immune effector cell that may contain the CAR described herein, the nucleic acid molecule, or the vector. In the present application, the immune effector cells may be mammalian cells. In the present application, the immune effector cells may be selected from T lymphocytes and natural killer (NK) cells.

[0195] Provided herein is a method for preparing immune effector cells, comprising introducing a vector as described herein into the immune effector cells. For example, the vector provided herein can be introduced into immune effector cells, such as T lymphocytes or natural killer (NK) cells. In certain embodiments, each or each cell may include one or a vector. In certain embodiments, each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) vectors. The vector is introduced into immune effector cells by methods known in the art. For example, immune effector cells can be transfected by retroviral vectors, and the viral genome with CAR molecules can be integrated into the host genome to ensure that the target gene is expressed long-term and stably. For another example, using a transposon, a plasmid carrying CAR (transposon) and a plasmid carrying a transposase are introduced into the target cell. For another example, CAR molecules can be added to the genome by gene editing (e.g., CRISPR / Cas9). In the present application, the vector with CAR molecules described herein can be introduced into the cells by methods known in the art, such as electroporation, liposome transfection, etc.

[0196] On the other hand, pharmaceutical compositions are provided herein, which may include the above-mentioned antibodies or their antigen-binding fragments, fusion proteins, nucleic acid molecules, carriers or immune effector cells, and pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or nonionic surfactants. In the present application, the pharmaceutical compositions may be formulated for oral administration, intravenous administration (e.g., intravenous injection, IV), intramuscular administration (e.g., intramuscular injection, IM), in situ administration at tumor site, suction, rectal administration, transdermal administration or administration by subcutaneous reservoir.

[0197] Pharmaceutical uses

[0198] Provided herein are uses of the above-mentioned antibodies or antigen-binding fragments thereof, fusion proteins, nucleic acid molecules, vectors, or immune effector cells for preparing a medicament for treating a disease or condition associated with ROR1 expression. In some embodiments, the disease or condition associated with ROR1 expression may be a tumor or cancer.

[0199] Also provided herein is a method for treating a disease, comprising administering the above-mentioned antibody or antigen-binding fragment thereof, fusion protein, nucleic acid molecule, vector, or immune effector cell to a subject suffering from or suspected of suffering from a disease or condition associated with ROR1 expression.

[0200] In some embodiments, the disease or disorder associated with the expression of ROR1 is a tumor or cancer.

[0201] In some embodiments, the tumor includes solid tumors such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and ovarian cancer.

[0202] Tumor immunotherapy by targeting ROR1 is a very useful treatment method. The inventors have developed new specific multi-antigen recognition epitope-targeted human ROR1 functional monoclonal nanoantibodies (single domain antibodies) and related chimeric antigen receptor T cell therapies, which specifically bind to different ROR1 antigen binding epitopes to reduce the possibility of drug resistance. They are used to inhibit the growth of ROR1-positive tumor cells and treat cancer diseases that express ROR1.

[0203] The present invention relates to antibodies and CAR-T molecules that are functionally directed against the human ROR1 target. The following examples describe embodiments of the invention in detail. Unless otherwise specified, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the methods and materials used in the examples described below are commercially available products. Those skilled in the art will understand that the methods and materials described below are illustrative only and should not be construed as limiting the scope of the invention.

[0204] Example 1: Obtaining human ROR1 single-domain antibody positive clone molecules

[0205] 1) Screening of lead antibody molecules specifically targeting human ROR1 using alpaca natural libraries

[0206] The alpaca natural library independently developed by Pengbo Bio was used for three rounds of liquid or solid phase screening with human ROR1-Biotin protein (fused with biotin protein tag, Biopsy, RO1-H821y, sequence reference Q01973-1), human ROR1 Frizzled-His domain protein (amino acid sequence of positions 165-305, fused with His protein tag, Biopsy, RO1-H5222, sequence reference Q01973-1) and human ROR1 Kringle-His domain protein (amino acid sequence of positions 308-395, fused with His protein tag, Biopsy, RO1-H5223, sequence reference Q01973-1) to obtain phage library eluate.

[0207] 2) Positive clone screening

[0208] Add the neutralized phage panning eluate to the prepared TG1 bacterial suspension, mix thoroughly, and infect the TG1 host bacteria at 37°C for 45 minutes. After sufficient infection, dilute the suspension in a gradient format and plate onto agar plates with the appropriate resistance profile. Incubate inverted at 37°C overnight. Prepare a sterile 96-well deep-well plate filled with 0.5 ml of 2YT medium (with 0.2% w / v glucose and 0.1 mg / ml ampicillin). Use a sterile pipette tip to pick a single colony from the plate and transfer it to the plate. Incubate at 37°C with shaking at 220 rpm overnight (16-18 hours). Transfer 0.05-0.1 ml of overnight cultured monoclonal bacterial solution to a freshly prepared sterile deep-well plate (containing 0.5 ml of 2YT medium containing a final concentration of 0.1 mg / ml ampicillin) and culture to an OD value of approximately 0.6-0.8 (under OD600 detection conditions). Add helper phage and shake to mix, then incubate at 37°C for 45 minutes. Then, add 0.5 ml of 2YT medium (containing 0.1 mg / ml ampicillin and kanamycin at a final concentration of 0.05 mg / ml) and culture overnight at 25°C with shaking at 220 rpm (16-18 hours). The overnight expressed bacterial solution was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant for subsequent testing of the selected monoclonal antibodies and the antigen protein human ROR1-His protein (fused His protein tag, Biopsies, RO1-H522y), human ROR1 Frizzled-His domain protein, and human ROR1 Kringle-His domain protein by ELISA binding assay and FACA binding assay against cellular antigens to obtain positive single domain antibody clones.

[0209] ELISA binding detection method: Indirect ELISA was used to evaluate the binding ability of phage-displayed antibodies in the supernatant for human ROR1-related proteins. ELISA plates were coated with 100 μl / well of CBS coating reagent at 1 μg / ml of recombinant human ROR1-His protein, human ROR1 Frizzled-His domain protein, and human ROR1 Kringle-His domain protein at 4°C overnight. 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 blocking solution was then discarded, and 50 μl of phage expression supernatant and 50 μl of 0.1% PBST were added to each plate, followed by incubation at room temperature for 2 hours. The plates were washed three times with PBST and incubated with 100 μl / well of horseradish peroxidase-conjugated goat anti-M13 phage antibody (Sino-Bio) at room temperature for 45 minutes. The plate was washed six times with PBST, then TMB colorimetric solution (GenScript) was added and incubated in the dark at room temperature for 10-15 minutes. The reaction was terminated by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader.

[0210] FACS assay: FACS binding assay was used to evaluate the ability of antibodies in the supernatant to bind to human ROR1 antigen expressed on the surface of CHO cell membranes. CHO cells expressing human ROR1 and negative control mother cells were collected for testing and washed three times with PBS. 2.5X10 5 Each test cell was incubated with 100 μl of the supernatant to be tested and 3.5 μg / ml of biotin-labeled anti-phage antibody at 4°C for 1 hour. The cells were then washed three times with PBS and 100 μl of iFluor-labeled streptavidin (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 BD Calibur FACS analyzer.

[0211] Example 2: Variable region sequencing of positive clones and recombinant production of monoclonal antibodies

[0212] Positive clones were selected based on the results of ELISA and FACS tests. The original colonies corresponding to the positive clones were cultured, and the expression plasmids were extracted, PCR and single clone Sanger sequencing were performed. Finally, 8 positive clones were obtained, as shown in Table 1.

[0213] Table 1. Positive clones and their predicted binding sites

[0214] The sequence of the antibody is shown below:

[0215] AHP15485 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1

[0216] AHP15485 CDR1 region amino acid sequence SEQ ID NO: 2

[0217] AHP15485 CDR2 region amino acid sequence SEQ ID NO: 3

[0218] AHP15485 CDR3 region amino acid sequence SEQ ID NO: 4

[0219] AHP15485 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 5

[0220] AHP15547 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 6

[0221] AHP15547 CDR1 region amino acid sequence SEQ ID NO: 7

[0222] AHP15547 CDR2 region amino acid sequence SEQ ID NO: 8

[0223] AHP15547 CDR3 region amino acid sequence SEQ ID NO: 9

[0224] AHP15547 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 10

[0225] AHP15580 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 11

[0226] AHP15580 CDR1 region amino acid sequence SEQ ID NO: 12

[0227] AHP15580 CDR2 region amino acid sequence SEQ ID NO: 13

[0228] AHP15580 CDR3 region amino acid sequence SEQ ID NO: 14

[0229] AHP15580 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 15

[0230] AHP15662 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 16

[0231] AHP15662 CDR1 region amino acid sequence SEQ ID NO: 17

[0232] AHP15662 CDR2 amino acid sequence SEQ ID NO: 18

[0233] AHP15662 CDR3 amino acid sequence SEQ ID NO: 19

[0234] AHP15662 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 20

[0235] AHP15768 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 21

[0236] AHP15768 CDR1 region amino acid sequence SEQ ID NO: 22

[0237] AHP15768 CDR2 region amino acid sequence SEQ ID NO: 23

[0238] AHP15768 CDR3 region amino acid sequence SEQ ID NO: 24

[0239] AHP15768 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 25

[0240] AHP15773 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 26

[0241] AHP15773 CDR1 region amino acid sequence SEQ ID NO: 27

[0242] AHP15773 CDR2 region amino acid sequence SEQ ID NO: 28

[0243] AHP15773 CDR3 amino acid sequence SEQ ID NO: 29

[0244] AHP15773 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 30

[0245] AHP15776 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 31

[0246] AHP15776 CDR1 region amino acid sequence SEQ ID NO: 32

[0247] AHP15776 CDR2 region amino acid sequence SEQ ID NO: 33

[0248] AHP15776 CDR3 region amino acid sequence SEQ ID NO: 34

[0249] AHP15776 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 35

[0250] AHP16026 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 36

[0251] AHP16026 CDR1 amino acid sequence SEQ ID NO: 37

[0252] AHP16026 CDR2 region amino acid sequence SEQ ID NO: 38

[0253] AHP16026 CDR3 region amino acid sequence SEQ ID NO: 39

[0254] AHP16026 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 40

[0255] 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.

[0256] HEK293-6E cells were transfected with the above plasmids and cultured in shake flasks at 37°C for 10 days. The supernatant was then collected for antibody purification. Prior to purification, the tubing 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 harvested cell culture supernatant was then diluted 1:1 with 2× 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 1× 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 exchanged into PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibodies were quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

[0257] The purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript). The gel was stained with Estain 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (GenScript).

[0258] Example 3: ELISA Binding Detection of Monoclonal Antibodies to Human ROR1 Recombinant Protein

[0259] Indirect ELISA was used to evaluate the binding ability of the above-mentioned purified antibodies to human ROR1-related recombinant proteins. ELISA plates (Nunc) were coated 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 at 4°C overnight. 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 blocking solution was then discarded, and 100 μl of purified antibody at 8 μg / ml (approximately 100 Nm) was added to the first well and diluted in a 3-fold gradient for a total of 11 test concentration gradients. The plates were then incubated at room temperature for 1 hour. The plates were washed three times with PBST and incubated with 100 μl / well of mouse anti-human IgG Fc fragment conjugated to horseradish peroxidase (GenScript) at 37°C for 0.5 hours. The plate was washed five times with PBST, then TMB colorimetric solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The reaction was terminated by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. As shown in Figures 1-3, the ELISA results of 8 positive recombinant antibody clones binding to ROR1-related antigen proteins, the EC values ​​of each antibody were 50 As shown in Table 2 below: Compared with the Yangshen antibody R11-scFv, these tested antibodies all reached or exceeded its antigen binding capacity.

[0260] Table 2. Purified antibodies and ROR1-related protein ELISA EC 50 Detection

[0261] Example 4: Binding of monoclonal antibodies to cell lines expressing human and mouse ROR1

[0262] The CHO cells expressing human and mouse ROR1 and the negative control cells were collected and washed three times with PBS. 2.5X10 5 Each test cell was incubated with 100 μl of purified antibody at 10 μg / ml at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of iFluor-conjugated goat anti-human IgG, Fcγ-specific antibody, 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 BD Calibur FACS reader. As shown in Figure 4, all antibodies bound to ROR1-expressing CHO-K1 / Human ROR1 stable cells and CHO-K1 / Mouse ROR1 stable cells, but not to the parent CHO cells.

[0263] Example 5: Humanized design and recombinant production of single-domain antibody molecules targeting human ROR1

[0264] The parent antibody structure is modeled by computer-assisted homology modeling software (MOE). A human natural germline sequence with high homology to the parent sequence is selected. The CDRs of the positive monoclonal antibody are grafted into the human natural germline sequence using CDR grafting technology to obtain a humanized chimeric antibody of the parent antibody. The different amino acid residues of the chimeric antibody and the parent antibody are compared. The key points that will affect the subsequent affinity are judged according to the classic residues, interaction loop region, core region, mutation hotspots, etc., and the appropriate sites are selected for combinatorial design to obtain the humanized antibody variable region amino acid sequence. The humanized antibody amino acid sequence is codon-optimized, and a heavy chain variable region DNA fragment containing the codon-optimized DNA fragment is synthesized and inserted into the pcDNA3.4-Fc (Human IgG1) expression vector to form an expression plasmid. The relevant expression plasmid is expressed according to the antibody recombinant method of Example 2, and the purification method is implemented to produce the humanized antibody.

[0265] AHP15485-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 41

[0266] AHP15485-VHH4 CDR1 region amino acid sequence SEQ ID NO: 2

[0267] AHP15485-VHH4 CDR2 region amino acid sequence SEQ ID NO: 3

[0268] AHP15485-VHH4 CDR3 region amino acid sequence SEQ ID NO: 4

[0269] AHP15485-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 42

[0270] AHP15662-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 43

[0271] AHP15662-VHH4 CDR1 region amino acid sequence SEQ ID NO: 17

[0272] AHP15662-VHH4 CDR2 region amino acid sequence SEQ ID NO: 18

[0273] AHP15662-VHH4 CDR3 region amino acid sequence SEQ ID NO: 19

[0274] AHP15662-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 44

[0275] AHP15773-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 45

[0276] AHP15773-VHH4 CDR1 region amino acid sequence SEQ ID NO: 27

[0277] AHP15773-VHH4 CDR2 region amino acid sequence SEQ ID NO: 28

[0278] AHP15773-VHH4 CDR3 region amino acid sequence SEQ ID NO: 29

[0279] AHP15773-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 46

[0280] Example 6: SPR Binding Detection of Humanized Antibodies to Human ROR1-Related Proteins

[0281] The affinity of the purified antibodies to human ROR1-related proteins was determined separately using the surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were immobilized on the sensor chip by Fc capture method. Human ROR1-related proteins were used as analytes. The dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated by the ratio of kd to ka. The antibodies were sorted according to their equilibrium dissociation constants, and humanized antibodies with no decrease in affinity or a decrease of no more than 3 times were selected for subsequent testing. The results of the SPR affinity determination are shown in Table 3 below, and the related sensor diagram is shown in Figure 5. After SPR affinity determination, the three groups of parental and humanized antibodies all had comparable affinity levels to their human ROR1 antigen proteins, and the equilibrium dissociation constants (KD) were all within 10 -7 within the range.

[0282] Table 3. Affinity determination of humanized single domain antibodies

[0283] Example 7: Binding of humanized antibodies to cell lines expressing human and mouse ROR1

[0284] The CHO cells expressing human and mouse ROR1 and the negative control cells were collected and washed three times with PBS. 2.5X10 5Each test cell was incubated with 100 μl of purified antibody at 10 μg / ml at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of Fluor-conjugated goat anti-human IgG, Fcγ-specific antibody, 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 BD Calibur FACS reader. As shown in Figure 6, all humanized antibodies bound to ROR1-expressing CHO-K1 / Human ROR1 stable cells and CHO-K1 / Mouse ROR1 stable cells, but not to the parental CHO cells, and their binding was comparable to that of the corresponding parental samples.

[0285] Example 8: Construction and detection of human ROR1 engineered cell lines

[0286] After synthesizing the ROR1 DNA ORF sequence (sequence from NP_005003.2, amino acid sequence as shown in SEQ ID NO: 47), the DNA fragment of ROR1 ORF was ligated with the vector backbone pLVX-Puromycin using Clone EZ (GenScript) technology and transformed into Escherichia coli competent cells to obtain the plasmid pLVX-ROR1-puromycin.

[0287] Lentivirus packaging: Using a three-plasmid system, pLVX-ROR1-puromycin and two auxiliary 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.

[0288] Infecting cells: Plate CHO-K1 cells (ECACC) into 6-well plates, add 3 mL of culture medium, and incubate overnight. Prior to infection, remove the cells from the refrigerator and rapidly thaw the virus in a 37°C water bath. Aspirate the culture medium from the cells, add 1 / 2 volume of fresh culture medium, and then add the original virus solution to the cells and mix thoroughly. After centrifugation for 0.5-1 hour, incubate the plate in a 37°C, 5% CO2 incubator for 24 hours. The day after infection (approximately 24 hours), aspirate the virus-containing culture medium and replace with fresh complete culture medium. Continue incubation at 37°C.

[0289] Puromycin resistance screening: Add 8 μg / ml puromycin (Thermo Fisher, Cat# A11138-03) to the cell culture medium. Replace the culture medium with complete puromycin every 2-3 days until the uninfected cells in the screening control group completely die under the selective pressure of puromycin. Continue screening until a stable cell line is obtained.

[0290] FACS detection of ROR1 expression: A portion of the obtained stable cells was transferred to a FACS tube and centrifuged to remove the supernatant. PE anti-human ROR1 Antibody (BioLegend, Cat#357804) was added and incubated at 4°C for 30 minutes. After 30 minutes, the supernatant was washed and resuspended in FACS buffer and loaded onto a BD FACS Celesta. TM ) were used to detect the expression level of ROR1.

[0291] Single clone selection: Dilute the cell pool to the minimum value in a 96-well plate. After 7 days, observe the 96-well plate under a microscope and mark the wells with single clones. Transfer the single clone to a 24-well plate and then expand it to a 6-well plate. After single clone expansion, verify the expression level of the CHO-K1 / ROR1 single clone using the above steps. The results of the best clone are shown in Figure 7. FACS analysis shows that CHO-K1 / ROR1 overexpresses ROR1.

[0292] Example 9: CAR vector structure

[0293] The present invention constructs a second-generation CAR vector targeting human ROR1, the structure of which is shown in Figure 8, and includes, for example, a ROR1 VHH antibody sequence, a CD28 transmembrane region, a 4-1BB intracellular region sequence, a CD3ζ sequence, and a tEGFR sequence as a transduction indicator protein. The amino acid sequences of the CARs (AHP15485 CAR, AHP15580 CAR, AHP15662 CAR, AHP15773 CAR, AHP15768 CAR, AHP16026 CAR, AHP15547 CAR, AHP15776 CAR) and R12 CAR of the present invention are shown in SEQ ID NOs: 56-64.

[0294] Example 10: Construction of human ROR1-CAR-Jurkat cells

[0295] In order to preliminarily verify the functional effect of ROR1 CAR, ROR1-CAR-Jurkat cells were constructed.

[0296] The Jurkat / NFAT-Luc cells were constructed as follows: Jurkat cells (ATCC) were cultured at a rate of 3 × 10 5 The cells were plated into 6-well plates and infected with NFAT-Luc virus concentrate. On the third day after infection (about 48 hours), resistance screening was performed using Hygromycin B (Thermo Fisher, Cat#10687-010) to obtain Jurkat / NFAT-Luc cell pools.

[0297] Produce ROR1 CAR concentrated virus. Lentivirus preparation refers to the lentiviral packaging technology in Example 8, Construction of ROR1 Engineering Cell Line.

[0298] Preparation of ROR1-CAR-Jurkat cells. Jurkat / NFAT-Luc cell pools were infected with ROR1 CAR concentrated virus at an MOI of 10. CAR expression was detected by FACS 72 hours after infection. As shown in Table 4, FACS detection of CAR transduction indicator protein EGFR expression (PE-EGFR Antibody, Novus, NBP2-75903PE) showed that the transduction efficiency was greater than 99%.

[0299] Table 4. Detection of positive rate of human ROR1-CAR-Jurkat cells

[0300] Example 11: Targeted human ROR1-CAR-Jurkat cells are specifically activated by target cells

[0301] In order to verify whether the ROR1-CAR constructed in the present invention can be specifically activated by ROR1-positive cells, ROR1-CAR Jurkat cells with 8 different ROR1 VHH sequences were constructed (SEQ ID NO: 56-63). R12 CAR-Jurkat cells constructed with R12 ScFv (Patent Publication No.: WO 2014 / 031687 A1) were used as a positive reference for the experiment (SEQ ID NO: 64). After co-incubation with CHO-K1 / ROR1 cells, the fluorescence signal value and IL-2 cytokine secretion were detected.

[0302] The activation effect of ROR1-CAR on Jurkat cells was evaluated based on the reporter gene method: the target cells CHO-K1 / ROR1 were collected, resuspended in complete culture medium F12K (Gibco, Cat#21127-022) + 10% FBS (Gibco, Cat#10091-148) and adjusted to a density of 2E5 cells / mL. Then, 50 μL of the target cell suspension was transferred to a 96-well plate, and the culture plate containing the target cells was transferred to an incubator at 37°C and 5% CO2 and incubated overnight. The next day, the effector ROR1-CAR Jurkat cells were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148), and then adjusted to a density of 4E5 cells / mL. The target cell culture plate that had been cultured overnight was taken out of the incubator, the previous medium was discarded, and 100 μL of the effector cell suspension was transferred. The culture plate was then transferred to an incubator at 37°C and 5% CO2 and incubated for 6 hours. The culture plate was removed and the detection reagent Fire-Lumi TM After incubation at room temperature for 5 minutes using a luciferase assay kit (GenScript, Cat#L00877C), luminescence signal values ​​were measured using a microplate reader (PHERAStar FSX, BMG). The results, as shown in Figure 10 and Table 5, show that among the eight sequences, sequences AHP15485, AHP15580, AHP15662, AHP15773, and AHP15776 exhibited relatively stronger activation effects on Jurkat cells, with signal ratios closer to those of the positive control, R12 ScFv.

[0303] Table 5. Signal ratios of ROR1-CAR Jurkat cell activation _ reporter gene method

[0304] The activation effect of ROR1-CAR on Jurkat cells was evaluated based on the cytokine IL-2 secretion level: the target cells CHO-K1 / ROR1 were collected, resuspended in its complete culture medium F12K (Gibco, Cat#21127-022) + 10% FBS (Gibco, Cat#10091-148) and adjusted to a density of 2E5 cells / mL. Then, 50 μL of the target cell suspension was transferred to a 96-well plate, and the culture plate containing the target cells was transferred to an incubator at 37°C and 5% CO2 and incubated overnight. The next day, the effector ROR1-CAR Jurkat cells were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148), then adjusted to a density of 4E5 cells / mL. The target cell culture plate, which had been incubated overnight, was removed from the incubator, the previous medium was discarded, and 100 μL of the effector cell suspension was transferred. The culture plate was then transferred to an incubator at 37°C and 5% CO2 and incubated for another 24 hours. The culture supernatant was collected and IL-2 cytokine secretion was detected using the Human IL2 kit (Cisbio, Cat#62HIL02PEH) according to its instructions. The signal value was detected by a microplate reader (PHERAStar FSX, BMG). The results are shown in FIG11 and Table 6 , which indicate that the molecular sequences AHP15485, AHP15580, AHP15662, AHP15773, and AHP15776 have relatively stronger T cell activation effects, and their signal ratios are closer to those of the positive control R12 ScFv.

[0305] Table 6. Ratio of IL-2 cytokine secretion levels in ROR1-CAR-Jurkat cells

[0306] ROR1-CAR-Jurkat cells had varying degrees of increased fluorescence signal values ​​and IL-2 secretion, indicating that the eight ROR1-CAR-Jurkat cells constructed by the present invention can be specifically activated by ROR1-positive cells, and suggesting that the molecular sequences AHP15485, AHP15580, AHP15662, AHP15773, and AHP15776 have relatively stronger T cell activation effects, and the fluorescence signal value detection results of the reporter gene method are consistent with the IL-2 cytokine secretion detection results.

[0307] Example 12: Construction and identification of ROR1-CAR-T cells

[0308] To further verify the function of each ROR1-CAR in primary T cells, ROR1-CAR-T cells with eight different ROR1 VHH sequences were constructed by lentiviral transfection. R12 CAR-T cells constructed with R12 ScFv served as a positive control for the experiment. The specific construction steps are as follows:

[0309] T cell activation: After thawing, frozen PBMCs (Sai ​​Li) were resuspended in AIM V complete medium (ThermoFisher, Cat#31035025) containing 300 U / ml rhIL-2 and counted. T cells were then activated at a 1:1 ratio of cells to CD3 / CD28 antibody magnetic beads (GenScript) (the activation time point was marked as D0, and the time points 1 day and 2 days after activation were marked as D1 and D2, respectively). The cells were incubated with magnetic beads for 24 h to obtain activated T cells.

[0310] Lentiviral T cell infection: Activated T cells were plated into 24-well plates at a cell density of 1E6 cells per well. CAR lentivirus (MOI = 10) was added to a total volume of 400 μl. 16 μl of the viral infection-enhancing reagent HiTransGP (Gene Gene, Cat#REVG005) was also added. 16 h after transfection, 1 ml of culture medium was added to each well for 72 h of culture to obtain ROR1-targeted CAR-T cells.

[0311] Flow cytometry to detect T cell proportion: 3 days after transfection (activation day 4), FACS was used to detect the CD3 ratio. The flow cytometry results are shown in Figure 9. The CD3 ratio is greater than 99%, indicating that the proportion of T cells in the cell population is greater than 99%.

[0312] CAR cell ratio was detected by flow cytometry: 3 days after transfection (activation day 4), CAR transduction efficiency was detected by FACS. The CAR positive rate results are shown in Table 7, and the CAR transfection efficiency was greater than 70%.

[0313] Table 7. ROR1-CAR-T positive rate detection

[0314] Example 13: In vitro killing experiment of ROR1-CAR-T cells on ROR1-positive target cells

[0315] Target CHO-K1 / ROR1 / Luc cells (Nanjing Probio, Cat#RD00949) were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148) to adjust the density to 1E5 cells / mL. 50 μL of the target cell suspension was then 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 according to the CAR positive rate of different transduction molecules (see Table 7). Then, 50 μL of ROR1-CAR-T cell suspension and 50 μL of 1% Triton X-100 (Beijing Bailingwei Technology Co., Ltd., Cat#993361) (as the maximum killing signal value) were transferred to a 96-well plate with target cells. The culture plate was then transferred to an incubator at 37°C and 5% CO2 for incubation for 24 hours. The culture plate was removed after incubation for 24 hours and the detection reagent Fire-Lumi TM After incubation at room temperature for 5 minutes using a luciferase assay kit (GenScript, Cat#L00877C), the luminescence signal value was detected using a microplate reader (PHERAStar FSX, BMG).

[0316] Killing calculation formula % Cytotoxicity = 100*(1-(RLU Experimental –RLU Min ) / (RLU UnT –RLU Min The cytotoxicity of ROR1-CAR-T cells transduced with different molecules was calculated. The results are shown in Figure 12 and Table 8. CAR T cells expressing the molecular sequences AHP15485, AHP15662, and AHP15773 exhibited the same degree of cytotoxicity as the positive control R12 CAR T (AHP15485% Cytotoxicity = 84.86%, AHP15662% Cytotoxicity = 96.69%, AHP15773% Cytotoxicity = 94.82%, and R12 CAR% Cytotoxicity = 90.28%).

[0317] RLU Experimental : Signal value of ROR1-CAR T cells + target cells CHO-K1 / ROR1 / Luc;

[0318] RLU UnT: Signal value of non-transduced T cells + target cells CHO-K1 / ROR1 / Luc. The target cells are not specifically killed by Car-T cells, and the signal value is the highest;

[0319] RLU Min : 1% Triton X-100 + target cell CHO-K1 / ROR1 / Luc signal value, target cells are completely lysed by Triton X-100, the signal value is the lowest (RLU in the killing calculation formula Min ).

[0320] Table 8. Cytotoxicity of ROR1-CAR-T cells against target cells CHO-K1 / ROR1 / Luc

[0321] Example 14: Cytokine release assay of ROR1-CAR-T cells on ROR1-positive target cells

[0322] Target CHO-K1 / ROR1 / Luc cells were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148) to adjust the density to 5E4 cells / mL. Then, 100 μL of the target cell suspension was transferred to a 96-well plate. ROR1-CAR T cells with different transduction molecules were collected and resuspended in complete medium 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148). The theoretical density was adjusted to 1E6 cells / mL according to the CAR positivity rate of different transduction molecules (see Table 7). Then, 100 μL of the ROR1-CAR-T suspension was transferred to the 96-well plate with target cells, and the culture plate was transferred to an incubator at 37°C, 5% CO2 and incubated for 24 h. After 24 hours of incubation, the culture plate was transferred out, and the culture supernatant was collected and the secretion of IL-2 and IFN-γ cytokines was detected by using the Human IL2 kit (Cisbio, Cat#62HIL02PEH) and Human IFN gamma kit (Cisbio, Cat#62HIFNGPEH) according to their instructions. The signal value was detected by a microplate reader (PHERAStar FSX, BMG). The results are shown in Figure 13 and Tables 9 and 10. Among the 8 molecular sequences, the CAR T cell cytokine secretion levels of molecular sequences AHP15485, AHP15662, and AHP15773 were relatively higher and comparable to the secretion level 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 = 107.49, R12 CAR S / B ratio = 107.63). ratio=265.67; comparison of IFN-γ secretion levels: AHP15485 S / B ratio=46.87, AHP15662 B ratio=48.45, AHP15773 S / B ratio=58.03, R12 CAR S / B ratio=51.33), and the detection results were consistent with the killing effect.

[0323] Table 9. IL-2 cytokine release by ROR1-CAR-T cells on target cells CHO-K1 / ROR1 / Luc

[0324] Table 10. IFN-γ cytokine release by ROR1-CAR-T cells on target cells CHO-K1 / ROR1 / Luc

[0325] Example 15: Affinity maturation and recombinant validation of humanized single domain antibodies

[0326] The humanized single-domain antibody is synthesized in a prokaryotic soluble expression vector from Pengbo for prokaryotic expression and binding verification. Once normal expression is confirmed, the prokaryotic soluble expression plasmid will serve as the template for the subsequent mutation library. GenScript orders the synthesis of a precise saturation mutation library for the CDR region of the single-domain antibody. Upon receipt of the synthetic library sample, an ELISA binding assay with the antigen protein is performed. Based on the ELISA results, clones with improved binding values ​​are selected for SPR ranking. Based on the dissociation constant ratio between the sample and the humanized single-domain antibody sample in the SPR binding assay, clones with significantly improved binding values ​​are selected for Sanger sequencing to determine the specific mutation site and mutated amino acid in the improved clones.

[0327] Based on the mutation status of the improved clones in the precise saturation mutation library, the second round of combinatorial mutations and related library construction primers were designed. The humanized single-domain antibody prokaryotic soluble expression plasmid was still used as a template to construct the combinatorial mutation library. The subsequent screening process was consistent with the precise saturation mutation library. Finally, the top 5 or 7 clones with the highest affinity improvement were selected for recombination verification. The humanized antibody AHP15485 was screened through affinity maturation to obtain five affinity-enhanced clones (AHF22016, AHF22018, AHF22013, AHP15485-VHH4-Variant1, AHP15485-VHH4-Variant2) for eukaryotic expression and purification and subsequent affinity multi-concentration testing, while seven clones (AHP15662-VHH4-Variant1, AHP15662-VHH4-Variant2, AHP15662-VHH4-Variant3, AHF21711, AHF21719, AHF21720, AHF21721) were obtained from AHP15662-VHH4. After eukaryotic recombinant expression and purification of the affinity-enhanced clone, the affinity of the purified antibody to human ROR1-related proteins was determined using a surface plasmon resonance (SPR) biosensor, Biacore T200 (GE Healthcare). The sequences of the affinity-enhanced clones are listed below. The specific affinity determination method is consistent with Example 6.

[0328] AHF22013 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 65

[0329] AHF22013 CDR1 region amino acid sequence: SEQ ID NO: 66

[0330] AHF22013 CDR2 region amino acid sequence: SEQ ID NO: 3

[0331] AHF22013 CDR3 region amino acid sequence: SEQ ID NO: 67

[0332] AHF22016 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 68

[0333] AHF22016 CDR1 region amino acid sequence: SEQ ID NO: 66

[0334] AHF22016 CDR2 region amino acid sequence: SEQ ID NO: 3

[0335] AHF22016 CDR3 region amino acid sequence: SEQ ID NO: 69

[0336] AHF22018 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 70

[0337] AHF22018 CDR1 region amino acid sequence: SEQ ID NO: 66

[0338] AHF22018 CDR2 region amino acid sequence: SEQ ID NO: 3

[0339] AHF22018 CDR3 region amino acid sequence: SEQ ID NO: 71

[0340] AHP15485-VHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 72

[0341] AHP15485-VHH4-Variant1 CDR1 region amino acid sequence: SEQ ID NO: 2

[0342] AHP15485-VHH4-Variant1 CDR2 region amino acid sequence: SEQ ID NO: 3

[0343] AHP15485-VHH4-Variant1 CDR3 region amino acid sequence: SEQ ID NO: 4

[0344] AHP15485-VHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 73

[0345] AHP15485-VHH4-Variant2 CDR1 region amino acid sequence: SEQ ID NO: 66

[0346] AHP15485-VHH4-Variant2 CDR2 region amino acid sequence: SEQ ID NO: 3

[0347] AHP15485-VHH4-Variant2 CDR3 region amino acid sequence: SEQ ID NO: 4

[0348] AHP15662-VHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 74

[0349] AHP15662-VHH4-Variant1 CDR1 region amino acid sequence: SEQ ID NO: 75

[0350] AHP15662-VHH4-Variant1 CDR2 region amino acid sequence: SEQ ID NO: 76

[0351] AHP15662-VHH4-Variant1 CDR3 region amino acid sequence: SEQ ID NO: 19

[0352] AHP15662-VHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 77

[0353] AHP15662-VHH4-Variant2 CDR1 region amino acid sequence: SEQ ID NO: 78

[0354] AHP15662-VHH4-Variant2 CDR2 region amino acid sequence: SEQ ID NO: 76

[0355] AHP15662-VHH4-Variant2 CDR3 region amino acid sequence: SEQ ID NO: 19

[0356] AHP15662-VHH4-Variant3 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 79

[0357] AHP15662-VHH4-Variant3 CDR1 region amino acid sequence: SEQ ID NO: 80

[0358] AHP15662-VHH4-Variant3 CDR2 region amino acid sequence: SEQ ID NO: 76

[0359] AHP15662-VHH4-Variant3 CDR3 region amino acid sequence: SEQ ID NO: 19

[0360] AHF21711 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 81

[0361] AHF21711 CDR1 region amino acid sequence: SEQ ID NO: 82

[0362] AHF21711 CDR2 region amino acid sequence: SEQ ID NO: 83

[0363] AHF21711 CDR3 region amino acid sequence: SEQ ID NO: 19

[0364] AHF21719 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 84

[0365] AHF21719 CDR1 region amino acid sequence: SEQ ID NO: 85

[0366] AHF21719 CDR2 region amino acid sequence: SEQ ID NO: 76

[0367] AHF21719 CDR3 region amino acid sequence: SEQ ID NO: 19

[0368] AHF21720 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 86

[0369] AHF21720 CDR1 region amino acid sequence: SEQ ID NO: 85

[0370] AHF21720 CDR2 region amino acid sequence: SEQ ID NO: 87

[0371] AHF21720 CDR3 region amino acid sequence: SEQ ID NO: 19

[0372] AHF21721 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 88

[0373] AHF21721 CDR1 region amino acid sequence: SEQ ID NO: 89

[0374] AHF21721 CDR2 region amino acid sequence: SEQ ID NO: 90

[0375] AHF21721 CDR3 region amino acid sequence: SEQ ID NO: 19

[0376] The results are shown in Tables 11-12 and Figures 14-15. After affinity maturation, the affinity of the humanized single-domain antibody AHP15485-VHH4 was increased to a maximum of 10 -9 level; the affinity of humanized single-domain antibody AHP15662-VHH4 was increased up to 10 -8 level.

[0377] Table 11. Affinity determination of AHP15485-VHH4 affinity matured antibodies

[0378] Table 12. Affinity determination of AHP15662-VHH4 affinity matured antibodies

[0379] The amino acid and nucleotide sequence information mentioned herein is as follows.

[0380] AHP15485 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1

[0381] AHP15485 CDR1 region amino acid sequence SEQ ID NO: 2

[0382] AHP15485 CDR2 region amino acid sequence SEQ ID NO: 3

[0383] AHP15485 CDR3 region amino acid sequence SEQ ID NO: 4

[0384] AHP15485 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 5

[0385] AHP15547 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 6

[0386] AHP15547 CDR1 region amino acid sequence SEQ ID NO: 7

[0387] AHP15547 CDR2 region amino acid sequence SEQ ID NO: 8

[0388] AHP15547 CDR3 region amino acid sequence SEQ ID NO: 9

[0389] AHP15547 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 10

[0390] AHP15580 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 11

[0391] AHP15580 CDR1 region amino acid sequence SEQ ID NO: 12

[0392] AHP15580 CDR2 region amino acid sequence SEQ ID NO: 13

[0393] AHP15580 CDR3 region amino acid sequence SEQ ID NO: 14

[0394] AHP15580 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 15

[0395] AHP15662 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 16

[0396] AHP15662 CDR1 region amino acid sequence SEQ ID NO: 17

[0397] AHP15662 CDR2 amino acid sequence SEQ ID NO: 18

[0398] AHP15662 CDR3 amino acid sequence SEQ ID NO: 19

[0399] AHP15662 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 20

[0400] AHP15768 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 21

[0401] AHP15768 CDR1 region amino acid sequence SEQ ID NO: 22

[0402] AHP15768 CDR2 region amino acid sequence SEQ ID NO: 23

[0403] AHP15768 CDR3 region amino acid sequence SEQ ID NO: 24

[0404] AHP15768 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 25

[0405] AHP15773 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 26

[0406] AHP15773 CDR1 region amino acid sequence SEQ ID NO: 27

[0407] AHP15773 CDR2 region amino acid sequence SEQ ID NO: 28

[0408] AHP15773 CDR3 amino acid sequence SEQ ID NO: 29

[0409] AHP15773 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 30

[0410] AHP15776 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 31

[0411] AHP15776 CDR1 region amino acid sequence SEQ ID NO: 32

[0412] AHP15776 CDR2 region amino acid sequence SEQ ID NO: 33

[0413] AHP15776 CDR3 region amino acid sequence SEQ ID NO: 34

[0414] AHP15776 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 35

[0415] AHP16026 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 36

[0416] AHP16026 CDR1 amino acid sequence SEQ ID NO: 37

[0417] AHP16026 CDR2 region amino acid sequence SEQ ID NO: 38

[0418] AHP16026 CDR3 region amino acid sequence SEQ ID NO: 39

[0419] AHP16026 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 40

[0420] AHP15485-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 41

[0421] AHP15485-VHH4 CDR1 region amino acid sequence SEQ ID NO: 2

[0422] AHP15485-VHH4 CDR2 region amino acid sequence SEQ ID NO: 3

[0423] AHP15485-VHH4 CDR3 region amino acid sequence SEQ ID NO: 4

[0424] AHP15485-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 42

[0425] AHP15662-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 43

[0426] AHP15662-VHH4 CDR1 region amino acid sequence SEQ ID NO: 17

[0427] AHP15662-VHH4 CDR2 region amino acid sequence SEQ ID NO: 18

[0428] AHP15662-VHH4 CDR3 region amino acid sequence SEQ ID NO: 19

[0429] AHP15662-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 44

[0430] AHP15773-VHH4 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 45

[0431] AHP15773-VHH4 CDR1 region amino acid sequence SEQ ID NO: 27

[0432] AHP15773-VHH4 CDR2 region amino acid sequence SEQ ID NO: 28

[0433] AHP15773-VHH4 CDR3 region amino acid sequence SEQ ID NO: 29

[0434] AHP15773-VHH4 single domain antibody heavy chain variable region DNA sequence: SEQ ID NO: 46

[0435] SEQ ID NO:47 ROR1 amino acid sequence

[0436] SEQ ID NO: 48 signal peptide

[0437] SEQ ID NO: 49 Linker (used in R12 CAR as a control)

[0438] SEQ ID NO:50 Hinge region

[0439] SEQ ID NO: 51 transmembrane region

[0440] SEQ ID NO:524-1BB

[0441] SEQ ID NO:53 CD3ζ

[0442] SEQ ID NO:54 T2A

[0443] SEQ ID NO:55 tEGFR

[0444] SEQ ID NO:56 AHP15485 CAR

[0445] SEQ ID NO:57 AHP15580 CAR

[0446] SEQ ID NO:58 AHP15662 CAR

[0447] SEQ ID NO:59 AHP15773 CAR

[0448] SEQ ID NO:60 AHP15768 CAR

[0449] SEQ ID NO:61 AHP16026 CAR

[0450] SEQ ID NO:62 AHP15547 CAR

[0451] SEQ ID NO:63 AHP15776 CAR

[0452] SEQ ID NO:64 R12 CAR

[0453] AHF22013 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 65

[0454] AHF22013 CDR1 region amino acid sequence SEQ ID NO: 66

[0455] AHF22013 CDR2 amino acid sequence SEQ ID NO: 3

[0456] AHF22013 CDR3 region amino acid sequence SEQ ID NO: 67

[0457] AHF22016 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 68

[0458] AHF22016 CDR1 region amino acid sequence SEQ ID NO: 66

[0459] AHF22016 CDR2 amino acid sequence SEQ ID NO: 3

[0460] AHF22016 CDR3 region amino acid sequence SEQ ID NO: 69

[0461] AHF22018 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 70

[0462] AHF22018 CDR1 region amino acid sequence SEQ ID NO: 66

[0463] AHF22018 CDR2 amino acid sequence SEQ ID NO: 3

[0464] AHF22018 CDR3 region amino acid sequence SEQ ID NO: 71

[0465] AHP15485-VHHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 72

[0466] AHP15485-VHHH4-Variant1 CDR1 region amino acid sequence SEQ ID NO: 2

[0467] AHP15485-VHHH4-Variant1 CDR2 region amino acid sequence SEQ ID NO: 3

[0468] AHP15485-VHHH4-Variant1 CDR3 region amino acid sequence SEQ ID NO: 4

[0469] AHP15485-VHHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 73

[0470] AHP15485-VHHH4-Variant2 CDR1 region amino acid sequence SEQ ID NO: 66

[0471] AHP15485-VHHH4-Variant2 CDR2 region amino acid sequence SEQ ID NO: 3

[0472] AHP15485-VHHH4-Variant2 CDR3 region amino acid sequence SEQ ID NO: 4

[0473] AHP15662-VHH4-Variant1 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 74

[0474] AHP15662-VHH4-Variant1 CDR1 region amino acid sequence SEQ ID NO: 75

[0475] AHP15662-VHH4-Variant1 CDR2 region amino acid sequence SEQ ID NO: 76

[0476] AHP15662-VHH4-Variant1 CDR3 region amino acid sequence SEQ ID NO: 19

[0477] AHP15662-VHH4-Variant2 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 77

[0478] AHP15662-VHH4-Variant2 CDR1 region amino acid sequence SEQ ID NO: 78

[0479] AHP15662-VHH4-Variant2 CDR2 region amino acid sequence SEQ ID NO: 76

[0480] AHP15662-VHH4-Variant2 CDR3 region amino acid sequence SEQ ID NO: 19

[0481] AHP15662-VHH4-Variant3 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 79

[0482] AHP15662-VHH4-Variant3 CDR1 region amino acid sequence SEQ ID NO:80

[0483] AHP15662-VHH4-Variant3 CDR2 region amino acid sequence SEQ ID NO: 76

[0484] AHP15662-VHH4-Variant3 CDR3 region amino acid sequence SEQ ID NO: 19

[0485] AHF21711 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 81

[0486] AHF21711 CDR1 region amino acid sequence SEQ ID NO:82

[0487] AHF21711 CDR2 region amino acid sequence SEQ ID NO:83

[0488] AHF21711 CDR3 region amino acid sequence SEQ ID NO: 19

[0489] AHF21719 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 84

[0490] AHF21719 CDR1 region amino acid sequence SEQ ID NO: 85

[0491] AHF21719 CDR2 region amino acid sequence SEQ ID NO: 76

[0492] AHF21719 CDR3 amino acid sequence SEQ ID NO: 19

[0493] AHF21720 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 86

[0494] AHF21720 CDR1 region amino acid sequence SEQ ID NO:85

[0495] AHF21720 CDR2 region amino acid sequence SEQ ID NO: 87

[0496] AHF21720 CDR3 region amino acid sequence SEQ ID NO: 19

[0497] AHF21721 single domain antibody heavy chain variable region amino acid sequence SEQ ID NO: 88

[0498] AHF21721 CDR1 region amino acid sequence SEQ ID NO: 89

[0499] AHF21721 CDR2 region amino acid sequence SEQ ID NO: 90

[0500] AHF21721 CDR3 region amino acid sequence SEQ ID NO: 19

Claims

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

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

3. The antibody or antigen-binding fragment thereof according to 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 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the amino acid sequence of the heavy chain variable region of the humanized antibody is selected from any one of the following: (1) a heavy chain variable region sequence represented by SEQ ID NO: 41 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (2) a heavy chain variable region sequence represented by SEQ ID NO: 43 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (3) a heavy chain variable region sequence represented by SEQ ID NO: 45 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (4) a heavy chain variable region sequence represented by SEQ ID NO: 72 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (5) a heavy chain variable region sequence represented by SEQ ID NO: 73 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (6) a heavy chain variable region sequence represented by SEQ ID NO: 74 or a heavy chain variable region sequence having at least 90% sequence identity thereto; (7) a heavy chain variable region sequence represented by SEQ ID NO: 77 or a heavy chain variable region sequence having at least 90% sequence identity thereto; and (8) The sequence shown in SEQ ID NO: 79 or a heavy chain variable region sequence 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. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the EC binding of the antibody to ROR1 protein, its Frizzled domain or its Kringle domain as determined by ELISA is 50 The value is no higher than 0.1μg / mL.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the KD value of the antibody binding to ROR1 protein, its Frizzled domain or its Kringle domain as determined by surface plasmon resonance is no greater than 10 -6 M, preferably not higher than 10 -7 M, more preferably not higher than 10 -8 M.

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

10. A fusion protein comprising at least one antigen-binding functional portion, wherein the antigen-binding functional portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. The fusion protein according to claim 10 , comprising at least two antigen-binding functional parts, wherein the two antigen-binding functional parts target the same or different antigen epitopes respectively.

12. The fusion protein according to claim 10 or 11, wherein: One of the two antigen-binding functional portions targets the Frizzled domain of the ROR1 protein, and the other of the two antigen-binding functional portions targets other parts of the ROR1 protein except the Frizzled domain; One of the two antigen-binding functional portions targets the Kringle domain of ROR1, and the other of the two antigen-binding functional portions targets other parts of the ROR1 protein except the Kringle domain; or One of the two antigen-binding functional portions targets the Kringle domain of ROR1, and the other of the two antigen-binding functional portions targets the Frizzled domain of ROR1.

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

14. A chimeric antigen receptor targeting ROR1 protein, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 1 to 9 or the fusion protein of any one of claims 10 to 13. The chimeric antigen receptor according to claim 14 , comprising the amino acid sequence shown in any one of SEQ ID NOs: 56-63.

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

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. The host cell according to claim 19, which is an immune effector cell and expresses the chimeric antigen receptor according to any one of claims 14 or 15.

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

22. A pharmaceutical composition comprising: 1) 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, or the host cell of any one of claims 19 to 21; and 2) Pharmaceutically acceptable carrier.

23. A method for treating a disease, comprising administering an effective amount 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, the host cell according to any one of claims 19 to 21, or the pharmaceutical composition according to claim 22 to a subject in need thereof. The method of claim 23 , wherein the disease is a tumor expressing ROR1 protein.

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

26. A kit for detecting ROR1 protein in a sample, wherein: The kit 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.

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 preparation of a medicament for treating tumors. The use according to claim 27 , wherein the disease is a tumor expressing ROR1 protein.

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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