Anti-ROR1 antibody and use thereof

Abstract

An anti-ROR1 antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the anti-ROR1 antibody or the antigen-binding fragment thereof, and a use of the anti-ROR1 antibody or the antigen-binding fragment thereof in the preparation of an antibody drug conjugate. The present disclosure further relates to an antibody drug conjugate containing the antibody or the antigen-binding fragment thereof, a preparation method for the antibody drug conjugate, and a use of the antibody drug conjugate.

Description

Anti-ROR1 antibodies and their uses

[0001] This application is based on the application with CN application number 202311056361.3 and application date August 21, 2023, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present disclosure belongs to the field of medicine. Specifically, the present disclosure relates to antibodies targeting ROR1, antibody-drug conjugates comprising the same, nucleic acid molecules encoding the same, and their use in the preparation of antibody-drug conjugates. The antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region, a light chain variable region, and a complementarity determining region disclosed herein. The present disclosure further relates to antibody-drug conjugates comprising the antibody or its antigen-binding fragment, and methods for preparing and using the same. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Cancer is one of the leading causes of illness and death in the world. As the population ages, the global cancer burden will continue to increase. The AACR report points out that by 2040, the total number of cancer patients worldwide will reach approximately 28 million, and an estimated 16.2 million patients will die from it.

[0005] Receptor tyrosine kinase-like orphan receptors (RORs) are members of the type I receptor tyrosine kinase (RTK) family and are a class of membrane receptors primarily comprised of ROR1 and ROR2. ROR1 is highly expressed during early embryonic development and plays a crucial role in various physiological processes, including regulating cell division, proliferation, and migration, and participating in the formation of organs such as nerves, bones, and blood vessels. During subsequent fetal development, ROR1 expression gradually decreases, and is essentially absent on the cell surfaces of normal pediatric and adult tissues (Semin Cancer Biol 2014 Vol. 29, 21-31). However, the expression of ROR1 is greatly increased in a variety of tumor cells, including solid tumor cells such as lung adenocarcinoma, breast cancer, ovarian cancer, melanoma, kidney cancer, gastric cancer, colorectal cancer, and pancreatic cancer (BMC Cancer. 2021 Nov 11; 21(1): 1199.) and certain blood malignancies such as chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), and the expression of ROR1 is closely related to the progression of the disease and the treatment effect (Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071; Front Oncol. 2021 May 28; 11: 680834). For example, in CLL patients, ROR1 is expressed in the mitochondrial membrane of the mitochondria, a type of mitochondrial membrane that is involved in the mitochondrial membrane of the mitochondria. + The proportion is over 90% (Blood. 2021 Jun 17; 137(24): 3365-3377.), and the expression level of ROR1 is closely related to the progression of CLL, making it a good biomarker for prognosis.

[0006] ROR1 plays a crucial role in various physiological processes by mediating signaling through the non-canonical Wnt signaling pathway, including regulating cell division, proliferation, migration, and chemotaxis, particularly Wnt5a. Wnt5a is a typical activator of the non-canonical Wnt signaling pathway, involved in phosphorylation of the NF-κB subunit p65, activating the NF-κB pathway in tumor cells, promoting cell migration and invasion, epithelial-mesenchymal transition (EMT), and cancer metastasis. As a receptor for Wnt5a, ROR1 participates in activating the NF-κB pathway in tumor cells. ROR1 plays a crucial role in the development of cancer stem cells and the EMT process. Cancer stem cells are cancer cells within tumors that exhibit more stem cell characteristics and are often more resistant to chemotherapy drugs. The EMT process transforms the cell morphology from epithelial to mesenchymal, making them more invasive and promoting cancer metastasis. In lung cancer, ROR1 is involved in the activation of proto-oncogenes (c-Src) and MET, leading to the inhibition of tumor cell apoptosis. ROR1 has also been found to be a scaffold protein for cavin-1 and caveolin-1, which can activate AKT in lung adenocarcinoma. In breast cancer, ROR1 promotes the activation of the PI3K / AKT pathway, and high ROR1 expression is associated with more severe disease progression. In summary, abnormal expression of ROR1 and related growth-promoting signaling events have been observed in many types of malignant tumors, making ROR1 an attractive therapeutic target for the development of anticancer drugs.

[0007] Given that ROR1 is highly expressed in various cancers, but absent or low in normal tissues, drug developers have developed a variety of ROR1-targeted anti-tumor therapeutic strategies. ADCs, among them, have made rapid progress and shown promising application prospects. By combining the high targeting properties of monoclonal antibodies with the high activity of cytotoxins in tumor tissue, ADCs are highly effective in killing tumor cells, have fewer side effects than chemotherapy drugs, and exhibit superior efficacy compared to traditional antibody-based cancer drugs. They have been dubbed "biological missiles" in the field of cancer treatment. To date, 14 ADCs have been approved for marketing worldwide, including seven for hematological malignancies and seven for solid tumors. Their targets include CD33, CD30, CD22, CD79b, HER2, Nectin-4, Trop-2, BCMA, CD19, and TF. Four of these ADCs have been launched in China.

[0008] Currently, no antibody-drug conjugate targeting ROR1 is on the market. Clinically-stage ROR1 ADCs include VLS-101 (WO2018237335), jointly developed by Merck & Co. (MSD) and VelosBio. VLS-101 consists of a monoclonal antibody targeting ROR1 and monomethyl auristatin E (MMAE). It is currently in Phase II clinical development for the treatment of patients with hematological malignancies and solid tumors. However, due to the limited therapeutic window of MMAE, it has only shown weak tumor inhibitory activity in preclinical efficacy models for solid tumors with low ROR1 expression. LCB71 / CS5001 (CN202010852078.1), an antibody-drug conjugate targeting ROR1, was jointly developed by CStone Pharmaceuticals and LegoChem Biosciences. LCB71 carries a tumor-activated pyrrolobenzodiazepine (PBD) protoxin. However, due to its own toxicity, PBD has a narrow safety window in the treatment of solid tumors (J Thorac Oncol. 2021 Sep; 16(9): 1429-1433.). In addition, NBE-002 (WO2017127664), an ADC candidate drug targeting ROR1 developed by Boehringer Ingelheim / NBE Therapeutics, couples PNU-159682 to the C-termini of the heavy and light chains of the antibody in a site-specific and quantitative manner through a specific reaction mediated by Sortase A. PNU-159682 has the same safety risks as PBD toxins.

[0009] ADC drugs are entering a period of rapid growth, with approvals increasing rapidly. As of December 2022, a total of 15 ADC drugs have been approved for marketing worldwide. Mylotarg, the world's first ADC drug, was approved for marketing in 2000 but withdrawn from the market in 2010 due to fatal liver damage. It was reintroduced in 2017 after optimizing its dosing regimen to improve efficacy and safety. Between 2001 and 2010, no ADC drugs were approved for marketing globally, marking a period of industry stagnation. With the continuous advancement of technology, ADC drugs are entering a period of concentrated marketing, with 10 approved to date. While new-generation ADCs, such as Enhertu, have achieved tremendous success, significant room for improvement remains, such as drug resistance and interstitial pneumonia side effects. Furthermore, unlike other ADC antigens, such as HER2, which has hundreds of thousands of copies on tumor cells, ROR1 is expressed at low levels in most tumor cells, with no more than 5,000 copies per cell. This necessitates an ADC molecule that is more effective at very low expression levels and has a wider safety window.

[0010] In addition, the mechanism of action of antibody-drug conjugates seems simple, but whether an antibody-drug conjugate can become a safe and effective drug is very complex and unpredictable, and depends on many factors. It can be seen that the research and development of ADC drugs requires a lot of experimental exploration and verification, and their safety and effectiveness cannot be predicted before the experiment.

[0011] Therefore, there is an urgent need in this field to provide more suitable new antibody-drug conjugates targeting ROR1, which can achieve efficient, simple and practical chemical preparation and conjugation, and improve the pharmaceutical properties and efficacy of existing antibody-drug conjugates targeting ROR1, such as comprehensively improving the stability of ADC molecules, increasing half-life, anti-tumor resistance, and therapeutic window.

[0012] Summary of the Invention

[0013] In order to solve the problems existing in the prior art, the first aspect of the present disclosure provides an antibody or an antigen-binding fragment thereof that can specifically bind to ROR1, comprising:

[0014] (1)(a) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0015] (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VH;

[0016] (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0017] (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0018] and / or

[0019] (b) the following three light chain variable region (VL) CDRs:

[0020] (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL;

[0021] (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0022] (vi) a VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL;

[0023] or

[0024] (2)(a) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0025] (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH;

[0026] (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and

[0027] (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0028] and / or

[0029] (b) the following three light chain variable region (VL) CDRs:

[0030] (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL;

[0031] (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0032] (vi) VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL.

[0033] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0034] In certain embodiments, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering systems.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0036] (1) the sequences of CDR1, CDR2, and CDR3 contained in VH as shown in SEQ ID NO: 37 or 41; and / or

[0037] The CDR1, CDR2 and CDR3 sequences contained in the VL as shown in SEQ ID NO: 38 or 42; or

[0038] (2) the sequences of CDR1, CDR2, and CDR3 contained in VH as shown in SEQ ID NO: 39; and / or

[0039] The CDR1, CDR2 and CDR3 sequences contained in VL are shown in SEQ ID NO:40.

[0040] In certain embodiments, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering systems.

[0041] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0042] (a) the following three heavy chain variable region (VH) CDRs:

[0043] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1,

[0044] (ii) a VH CDR2 consisting of the sequence of SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2, or consisting of the sequence of SEQ ID NO: 45, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 45, and

[0045] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3;

[0046] and / or

[0047] (b) the following three light chain variable region (VL) CDRs:

[0048] (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 4, or a sequence consisting of the following sequence: SEQ ID NO: 46, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 46,

[0049] (v) a VL CDR2 consisting of SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5, and

[0050] (vi) VL CDR3 consisting of SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6.

[0051] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0052] (a) the following three heavy chain variable region (VH) CDRs:

[0053] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1,

[0054] (ii) a VH CDR2 consisting of the sequence of SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2, or consisting of the sequence of SEQ ID NO: 45, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 45, and

[0055] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3;

[0056] and / or

[0057] (b) the following three light chain variable region (VL) CDRs:

[0058] (iv) a VL CDR1 consisting of SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 4,

[0059] (v) a VL CDR2 consisting of SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5, and

[0060] (vi) VL CDR3 consisting of SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6.

[0061] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0062] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and a VH CDR3 as set forth in SEQ ID NO: 3; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0063] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 45; and a VH CDR3 as set forth in SEQ ID NO: 3; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0064] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and a VH CDR3 as set forth in SEQ ID NO: 3; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 46; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0065] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 45; and a VH CDR3 as set forth in SEQ ID NO: 3; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 46; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0066] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and a VH CDR3 as set forth in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0067] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 45; and a VH CDR3 as set forth in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0068] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and a VH CDR3 as set forth in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 46; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0069] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 45; and a VH CDR3 as set forth in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 46; a VL CDR2 as set forth in SEQ ID NO: 5; and a VL CDR3 as set forth in SEQ ID NO: 6.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0071] (a) the following three heavy chain variable region (VH) CDRs:

[0072] (i) a VH CDR1 consisting of SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7,

[0073] (ii) a VH CDR2 consisting of SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8, and

[0074] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9;

[0075] and / or

[0076] (b) the following three light chain variable region (VL) CDRs:

[0077] (iv) a VL CDR1 consisting of the sequence of SEQ ID NO: 10, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10, or consisting of the sequence of SEQ ID NO: 47, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 47,

[0078] (v) a VL CDR2 consisting of SEQ ID NO: 11, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 11, and

[0079] (vi) VL CDR3 consisting of SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12.

[0080] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0081] (a) the following three heavy chain variable region (VH) CDRs:

[0082] (i) a VH CDR1 consisting of SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7,

[0083] (ii) a VH CDR2 consisting of SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8, and

[0084] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9;

[0085] and / or

[0086] (b) the following three light chain variable region (VL) CDRs:

[0087] (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10,

[0088] (v) a VL CDR2 consisting of SEQ ID NO: 11, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 11, and

[0089] (vi) VL CDR3 consisting of SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12.

[0090] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0091] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 7; a VH CDR2 as set forth in SEQ ID NO: 8; and a VH CDR3 as set forth in SEQ ID NO: 9; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 10; a VL CDR2 as set forth in SEQ ID NO: 11; and a VL CDR3 as set forth in SEQ ID NO: 12.

[0092] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 7; a VH CDR2 as set forth in SEQ ID NO: 8; and a VH CDR3 as set forth in SEQ ID NO: 9; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 47; a VL CDR2 as set forth in SEQ ID NO: 11; and a VL CDR3 as set forth in SEQ ID NO: 12.

[0093] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 7; a VH CDR2 as set forth in SEQ ID NO: 8; and a VH CDR3 as set forth in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 10; a VL CDR2 as set forth in SEQ ID NO: 11; and a VL CDR3 as set forth in SEQ ID NO: 12.

[0094] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 7; a VH CDR2 as set forth in SEQ ID NO: 8; and a VH CDR3 as set forth in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 47; a VL CDR2 as set forth in SEQ ID NO: 11; and a VL CDR3 as set forth in SEQ ID NO: 12.

[0095] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0096] (a) the following three heavy chain variable region (VH) CDRs:

[0097] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 13,

[0098] (ii) a VH CDR2 consisting of SEQ ID NO: 14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 14, and

[0099] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 15;

[0100] and / or

[0101] (b) the following three light chain variable region (VL) CDRs:

[0102] (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 16,

[0103] (v) a VL CDR2 consisting of SEQ ID NO: 17, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 17, and

[0104] (vi) VL CDR3 consisting of SEQ ID NO: 18, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 18.

[0105] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0106] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 13; a VH CDR2 as set forth in SEQ ID NO: 14; and a VH CDR3 as set forth in SEQ ID NO: 15; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 16; a VL CDR2 as set forth in SEQ ID NO: 17; and a VL CDR3 as set forth in SEQ ID NO: 18.

[0107] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 13; a VH CDR2 as set forth in SEQ ID NO: 14; and a VH CDR3 as set forth in SEQ ID NO: 15; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 16; a VL CDR2 as set forth in SEQ ID NO: 17; and a VL CDR3 as set forth in SEQ ID NO: 18.

[0108] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0109] (a) the following three heavy chain variable region (VH) CDRs:

[0110] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 19,

[0111] (ii) a VH CDR2 consisting of SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 20, and

[0112] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 21;

[0113] and / or

[0114] (b) the following three light chain variable region (VL) CDRs:

[0115] (iv) a VL CDR1 consisting of SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 22,

[0116] (v) a VL CDR2 consisting of SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 23, and

[0117] (vi) VL CDR3 consisting of SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24.

[0118] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0119] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 19; a VH CDR2 as set forth in SEQ ID NO: 20; and a VH CDR3 as set forth in SEQ ID NO: 21; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 22; a VL CDR2 as set forth in SEQ ID NO: 23; and a VL CDR3 as set forth in SEQ ID NO: 24.

[0120] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 19; a VH CDR2 as set forth in SEQ ID NO: 20; and a VH CDR3 as set forth in SEQ ID NO: 21; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 22; a VL CDR2 as set forth in SEQ ID NO: 23; and a VL CDR3 as set forth in SEQ ID NO: 24.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0122] (a) the following three heavy chain variable region (VH) CDRs:

[0123] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 25,

[0124] (ii) a VH CDR2 consisting of SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 26, and

[0125] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 27;

[0126] and / or

[0127] (b) the following three light chain variable region (VL) CDRs:

[0128] (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 28,

[0129] (v) a VL CDR2 consisting of SEQ ID NO: 29, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 29, and

[0130] (vi) VL CDR3 consisting of SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 30.

[0131] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0132] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 25; a VH CDR2 as set forth in SEQ ID NO: 26; and a VH CDR3 as set forth in SEQ ID NO: 27; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 28; a VL CDR2 as set forth in SEQ ID NO: 29; and a VL CDR3 as set forth in SEQ ID NO: 30.

[0133] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 25; a VH CDR2 as set forth in SEQ ID NO: 26; and a VH CDR3 as set forth in SEQ ID NO: 27; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 28; a VL CDR2 as set forth in SEQ ID NO: 29; and a VL CDR3 as set forth in SEQ ID NO: 30.

[0134] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0135] (a) the following three heavy chain variable region (VH) CDRs:

[0136] (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 31,

[0137] (ii) a VH CDR2 consisting of SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 32, and

[0138] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 33, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 33;

[0139] and / or

[0140] (b) the following three light chain variable region (VL) CDRs:

[0141] (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 34, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 34,

[0142] (v) a VL CDR2 consisting of SEQ ID NO: 35, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 35, and

[0143] (vi) VL CDR3 consisting of SEQ ID NO: 36, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 36.

[0144] In certain embodiments, the substitutions described in any one of (i)-(vi) above are conservative substitutions.

[0145] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 31; a VH CDR2 as set forth in SEQ ID NO: 32; and a VH CDR3 as set forth in SEQ ID NO: 33; and / or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO: 34; a VL CDR2 as set forth in SEQ ID NO: 35; and a VL CDR3 as set forth in SEQ ID NO: 36.

[0146] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO:31; a VH CDR2 as set forth in SEQ ID NO:32; and a VH CDR3 as set forth in SEQ ID NO:33; and the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as set forth in SEQ ID NO:34; a VL CDR2 as set forth in SEQ ID NO:35; and a VL CDR3 as set forth in SEQ ID NO:36.

[0147] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0148] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0149] (i) the sequence shown in SEQ ID NO: 37;

[0150] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 37; or

[0151] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 37;

[0152] and

[0153] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0154] (iv) the sequence shown in SEQ ID NO: 38;

[0155] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 38; or

[0156] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:38.

[0157] In certain embodiments, the substitutions described in (ii) or (v) above are conservative substitutions.

[0158] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0159] a heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO:37, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in SEQ ID NO:37, and a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO:38, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in SEQ ID NO:38.

[0160] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0161] The VH has the sequence shown in SEQ ID NO:37 and the VL has the sequence shown in SEQ ID NO:38.

[0162] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0163] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0164] (i) the sequence shown in SEQ ID NO: 39;

[0165] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 39; or

[0166] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 39;

[0167] and

[0168] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0169] (iv) the sequence shown in SEQ ID NO: 40;

[0170] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 40; or

[0171] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:40.

[0172] In certain embodiments, the substitutions described in (ii) or (v) above are conservative substitutions.

[0173] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0174] a heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO:39, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in SEQ ID NO:39, and a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO:40, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in SEQ ID NO:40.

[0175] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0176] The VH has the sequence shown in SEQ ID NO:39 and the VL has the sequence shown in SEQ ID NO:40.

[0177] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0178] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0179] (i) the sequence shown in SEQ ID NO: 41;

[0180] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 41; or

[0181] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:41;

[0182] and

[0183] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0184] (iv) the sequence shown in SEQ ID NO: 42;

[0185] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 42; or

[0186] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:42.

[0187] In certain embodiments, the substitutions described in (ii) or (v) above are conservative substitutions.

[0188] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0189] a heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO:41, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:41, and a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO:42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:43.

[0190] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0191] The VH has the sequence shown in SEQ ID NO:41 and the VL has the sequence shown in SEQ ID NO:42.

[0192] In certain embodiments, the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is humanized.

[0193] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.

[0194] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), and / or a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in the amino acid sequence encoded by a human light chain germline antibody gene).

[0195] In certain embodiments, the heavy chain framework region and / or light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from a human residue to a murine residue.

[0196] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0197] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0198] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

[0199] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a mouse antibody, a rabbit antibody, a humanized antibody, a fully human antibody, a chimeric antibody (e.g., a human-mouse chimeric antibody), a bispecific antibody, a multispecific antibody, a single-chain antibody, a dAb, a complementarity determining region fragment, Fv, a single-chain Fv (scFv), Fd, Fab, Fab', and F(ab')2.

[0200] In certain embodiments, the monoclonal antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, such as a human antibody.

[0201] In certain embodiments, the antibody or antigen-binding fragment thereof has a mass fraction less than or equal to 1.0×10 -8 M's K D Binds human or monkey ROR1.

[0202] The second aspect of the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region, or encoding the antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region.

[0203] The third aspect of the present disclosure provides a vector comprising the isolated nucleic acid molecule.

[0204] The fourth aspect of the present disclosure provides a host cell comprising the nucleic acid molecule or the vector.

[0205] The fifth aspect of the present disclosure provides the use of the antibody or its antigen-binding fragment in the preparation of an antibody-drug conjugate, or in the preparation of a kit for diagnosing a disease, especially a ROR1-mediated disease or condition, or in the preparation of a medicament for preventing and / or treating a disease, especially a ROR1-mediated disease or condition.

[0206] The present disclosure also provides the antibody or antigen-binding fragment thereof, which is used as an antibody-drug conjugate, or for diagnosing, preventing and / or treating diseases, especially ROR1-mediated diseases or conditions.

[0207] The present disclosure also provides a method for diagnosing, preventing and / or treating a disease, particularly a disease or condition mediated by ROR1, comprising administering an effective amount of the antibody or antigen-binding fragment thereof to a subject in need thereof.

[0208] The sixth aspect of the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in formula (I),

[0209] in:

[0210] Q1-L1-L2-L3 is a linker,

[0211] Ab is the antibody or antigen-binding fragment thereof, which is coupled to Q1 in the linker via the S atom in the antibody or antigen-binding fragment thereof,

[0212] T is a drug unit or a diagnostic agent unit, which is coupled to L3 in the linker through the N atom or O atom in the drug unit or the diagnostic agent unit,

[0213] m is a number between 1 and 9, for example, m is a number between 2 and 9, for example, about 4, about 5, about 6, about 7, about 8, or about 9,

[0214] Q1 is selected from: Replaced Replaced Replaced and replaced

[0215] L1 is -L 1a -C(=O)-, where L 1a is selected from the group consisting of: an alkylene group, a polyethylene glycol group, an alkenylene group, an alkynylene group, an alicyclic group, an alicyclic heterocyclic group, an arylene group, a heteroarylene group, a substituted alkylene group, a substituted polyethylene glycol group, a substituted alkenylene group, a substituted alkynylene group, a substituted alicyclic group, a substituted alicyclic heterocyclic group, a substituted arylene group, and a substituted heteroarylene group,

[0216] L2 is a polypeptide residue or a substituted polypeptide residue,

[0217] L3 is or replaced

[0218] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Q1 is selected from:

[0219] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Q1 is

[0220] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L 1a Selected from: alkylene group, polyethylene glycol group, alkenylene group, alkynylene group, alicyclic group.

[0221] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L 1a -(CH2) n1 -, n1 is a number between 1 and 10, such as a number between 1 and 8, a number between 1 and 6, a number between 1 and 4. In certain embodiments, n1 is 1, 2, 3, 4, 5, 6, 7 or 8.

[0222] In certain embodiments, L1 is -(CH2)5-C(=O)-.

[0223] In certain embodiments, in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, L2 and / or L3 may be substituted with CH2NHCH3, a polysarcosine residue, a glycosylated polyethylene glycol fragment, a substituted polysarcosine residue or a substituted glycosylated polyethylene glycol fragment.

[0224] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide residue comprises an amino acid selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine.

[0225] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide residues are selected from: phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) (SEQ ID NO: 48).

[0226] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the polypeptide residues are selected from: valine-alanine (Val-Ala), valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0227] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L3 is

[0228] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L3 is substituted with -CH2NHCH3

[0229] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L3 is substituted with a polysarcosine residue, a glycosylated polyethylene glycol fragment, a substituted polysarcosine residue or a substituted glycosylated polyethylene glycol fragment The polysarcosine residue is connected to the benzene ring through the structural unit -X-.

[0230] The structural unit -X- is or replaced

[0231] The substituted polysarcosine residue is wherein n2 is a number between 4 and 18, for example, a number between 4 and 12, and R is selected from the group consisting of: C1-C6 alkyl, C1-C6 cycloalkyl, and C1-C6 alkoxy,

[0232] The substituted glycosylated polyethylene glycol fragment is Here, n3 is a number between 4 and 18, for example, a number between 4 and 12.

[0233] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L3 is substituted by a polysarcosine residue, a glycosylated polyethylene glycol fragment Among them, polysarcosine residues and glycosylated polyethylene glycol are connected to the benzene ring through the structural unit -X-.

[0234] The structural unit -X- is

[0235] The substituted polysarcosine residue is wherein n2 is a number between 4 and 18, for example, a number between 4 and 12, and R is selected from the group consisting of: C1-C6 alkyl, C3-C6 cycloalkyl, and C1-C6 alkoxy,

[0236] The substituted glycosylated polyethylene glycol fragment is wherein n3 is a number between 4 and 18, for example, a number between 4 and 12. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n2 is 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n2 is 10.

[0237] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein R is selected from: C1-C4 alkyl and C3-C6 cycloalkyl. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein R is selected from: C1-C4 alkyl. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein R is selected from: methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein R is methyl.

[0238] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n3 is 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n3 is 8 or 11.

[0239] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L3 is

[0240] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein m is a number between 7 and 9, for example, 7.5-9, for example, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0.

[0241] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the drug unit is selected from:

[0242] (i) Tubulin inhibitors, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin or its analogs (e.g., tubulysin A, tubulysin B, tubulysin D, tubulysin H, tubulysin U, tubulysin V), eribulin or its analogs, vinblastine or its analogs (e.g., vincristine, vindesine, vinblastine, DAVLBH), Aur0101;

[0243] (ii) Camptothecin topoisomerase inhibitors, such as exatecan (CAS 171335-80-1), DXd (CAS 1599440-33-1), 7-ethyl-10-hydroxycamptothecin (SN38, CAS 86639-52-3), belotecan (CAS 213819-48-8), (4-NH2)-exatecan (AZD'0132, CAS 2495742-21-5), 7-MAD-MDCPT (CAS 765871-81-6), and 7-aminomethyl-10-methyl-11-fluoro camptothecin (CAS 2378616-23-8);

[0244] (iii) Immune activators, such as TLR7 / 8 agonists and STING agonists.

[0245] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Q1-L1-L2-L3-T is selected from:

[0246] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein -Q1-L1-L2-L3-T is selected from:

[0247] In certain embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from:

[0248] wherein m and Ab are as defined in the present disclosure.

[0249] A seventh aspect of the present disclosure provides a method for preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, comprising:

[0250] Reduction of Ab to obtain Ab';

[0251] Ab' is coupled with the compound represented by formula LD to obtain an antibody-drug conjugate represented by formula (I).

[0252] wherein: Ab, Q1, L1, L2, L3, T and m are as defined in the present disclosure.

[0253] In certain embodiments, the compound of formula LD is selected from compounds LD-3 to LD-11.

[0254] A typical preparation method includes: diluting the antibody stock solution to 2-30 mg / mL with reaction buffer, adding a 140-200-fold molar excess of dithiothreitol (DTT), or adding a 6.0-20-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stirring the reaction solution at 35-39°C for 2-48 hours. The reaction buffer may be a buffer prepared in the following proportions: 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate-citric acid / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6-9; 50 mM boric acid-borax / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6-9; PBS / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6-9; PBS / EDTA (ethylenediaminetetraacetic acid), pH 6-9; and a buffer containing 50 mM sodium chloride and 2 mM EDTA in phosphate buffer (50 mM), pH 6-9 (e.g., pH 7.0).

[0255] Cool the reaction mixture to 0-10°C. If DTT reduction is used, remove excess DTT by desalting or ultrafiltration after the reduction reaction is complete. Add the substituted maleimide compound (pre-dissolved in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or diethylacetamide (DMA) at a concentration of 10 mg / ml). Ensure that the volume of organic solvent in the total reaction solution does not exceed 15%. Stir the coupling reaction at 0-37°C for 2-4 hours. If TCEP reduction is used, the substituted maleimide compound can be added directly for coupling without removing the remaining TCEP.

[0256] The coupling reaction mixture was purified by filtration using a desalting column with histidine-acetate buffer containing sodium chloride or a phosphate buffer containing sodium chloride, centrifuged, and the flow-through fraction was collected.

[0257] The resulting antibody-drug conjugates have relatively uniform DAR values. When using the various substituted maleimide linkers (linker fragments) disclosed herein, ADC product homogeneity is very high (typically, DAR-dominant products (e.g., DAR values ​​of approximately 4, 5, 6, 7, 8, or 9) account for at least 60%, at least 70%, at least 80%, at least 90%, or even higher, of all ADCs). For ADCs with varying DARs, further separation and purification using, but not limited to, hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC) can be performed to obtain even more uniform samples.

[0258] The eighth aspect of the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

[0259] In certain embodiments, the antibody-drug conjugate is present in the pharmaceutical composition in an amount effective for treating a disease. The antibody-drug conjugate can be administered as a single active pharmaceutical ingredient or in combination with other drugs, such as other anticancer drugs. In some embodiments, the pharmaceutical composition further contains other drugs, such as other anticancer drugs. The other anticancer drugs include but are not limited to anti-PD-1 antibodies, anti-PDL-1 antibodies, and / or anti-CTLA-4 antibodies.

[0260] In certain embodiments, the antibody-drug conjugate or a pharmaceutical composition comprising the same is administered in an effective amount or effective dose, for example, in the range of 1 ng / kg to about 20-100 mg / kg body weight per day, and can be administered once, twice, three times or more per day for 1-4 weeks or longer.

[0261] The antibody-drug conjugate or the pharmaceutical composition comprising the same can be administered via any conventional route, such as intravenous, intraarterial, intramuscular, intraperitoneal administration, and the like.

[0262] The ninth aspect of the present disclosure provides the use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a medicament for preventing and / or treating a disease, particularly a ROR1-mediated disease or condition, or in preparing a medicament for treating and / or preventing a tumor, or in preparing a kit for diagnosing a disease, particularly a ROR1-mediated disease or condition.

[0263] The present disclosure also provides the antibody-drug conjugate or a pharmaceutically acceptable salt thereof or the pharmaceutical composition, which is used for diagnosing, preventing and / or treating diseases, especially ROR1-mediated diseases or conditions, or for treating and / or preventing tumors.

[0264] The tenth aspect of the present disclosure provides a kit or composition comprising the antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule, the vector, the host cell, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0265] In certain embodiments, the kit comprises the anti-ROR1 antibody or antigen-binding fragment thereof, and instructions for use. The kit can be used to diagnose a disease, particularly a disease or condition mediated by ROR1.

[0266] Such a kit may, for example, comprise a disposable test device configured to generate a detectable signal associated with the presence or amount of ROR1 in a biological sample. Alternatively, such a kit may be formulated for determination in a clinical analyzer that does not utilize a disposable test device. Preferably, the kit is an in vitro diagnostic agent. As used herein, the term "in vitro diagnostic" refers to a medical device, which is a reagent, reagent product, calibrator, control material, kit, instrument, device, equipment or system, whether used alone or in combination, intended by the manufacturer for in vitro examination of specimens from the human body (including blood and tissue donations), specifically provided or noted for providing information about physiological or pathological states or about congenital anomalies or determining safety and compatibility with potential recipients, or monitoring therapeutic measures.

[0267] The eleventh aspect of the present disclosure provides a method for preventing and / or treating a disease, particularly a ROR1-mediated disease or condition, comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, or the pharmaceutical composition.

[0268] A twelfth aspect of the present disclosure provides a method for diagnosing or prognosing a disease (particularly a disease or condition mediated by ROR1), comprising:

[0269] Collect a tissue sample of interest from a subject,

[0270] The tissue sample is contacted with the antibody or antigen-binding fragment thereof.

[0271] If a certain amount of ROR1 is detected, the subject may be diagnosed with cancer. Further, if the detected ROR1 expression increases / decreases, it indicates that the cancer is progressing / improving.

[0272] In certain embodiments, the ROR1-mediated disease or disorder is a cancer that is highly expressed in ROR1. In certain embodiments, the cancer that is highly expressed in ROR1 includes, but is not limited to, head and neck squamous cell carcinoma, lung cancer (e.g., non-small cell lung cancer, including lung adenocarcinoma and lung squamous cell carcinoma), urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, melanoma, leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL)), malignant lymphoma (e.g., mantle cell lymphoma (MCL)), plasmacytoma, myeloma, sarcoma.

[0273] In certain embodiments, the tumor is selected from the group consisting of squamous cell carcinoma of the head and neck, lung cancer, urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, melanoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma.

[0274] In certain embodiments, the tumor or cancer is selected from head and neck squamous cell carcinoma, pancreatic cancer, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma, and colorectal cancer.

[0275] In certain embodiments, the tumor or cancer is selected from the group consisting of head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung cancer, and triple-negative breast cancer.

[0276] In certain embodiments, the non-small cell lung cancer is selected from the group consisting of lung adenocarcinoma and lung squamous cell carcinoma.

[0277] Other features and advantages of the present disclosure will be set forth in the subsequent description and, in part, will become apparent from the description or will be understood through practice of the present disclosure. Other advantages of the present disclosure can be achieved and obtained through the schemes described in the description. The contents of all references, Genbank entries, patents, and published patent applications cited in this disclosure are expressly incorporated herein by reference.

[0278] Definition of terms

[0279] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0280] As used herein, the term "and / or" should be considered as a specific disclosure of each of two or more specified features or elements, with or without the other features or elements. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0281] It will be understood that wherever aspects are described using the language "comprising" or "including," similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0282] As used herein, the term "about" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies at an acceptable level in the art. In some embodiments, such variation can be as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values.

[0283] As used in this article, the term "ROR1" (receptor tyrosine kinase-like orphan receptor 1) is a type I transmembrane protein that includes an extracellular part, a transmembrane part, and a cytoplasmic region, and belongs to the receptor tyrosine kinase-like orphan receptor (ROR) family. It is now known that ROR family members are receptors for Wnt family signaling molecules Wnt5a / b and Wnt16, with Wnt5a as the main ligand. ROR1 mediates signal transduction of the non-canonical Wnt signaling pathway (non-canonical Wnt pathways) by binding to the ligand Wnt5a. This signal is involved in a variety of cellular physiological processes and the occurrence and development of various cancers. ROR1 is highly expressed during early embryonic development and is involved in regulating cell division, proliferation, migration, etc., and is involved in the generation of organs such as nerves, bones, and blood vessels. As the fetus develops, the expression of ROR1 gradually decreases. In children and adults, ROR1 is expressed at very low or no expression in almost all normal tissues. In contrast to normal tissues, ROR1 is expressed in a variety of cancers, including hematological cancers such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and solid tumors (ovarian, breast, prostate, lung, melanoma, and colorectal cancer, among others).

[0284] High expression of ROR1 is usually determined by comparing its expression levels in cancer tissues and normal tissues. Usually, this is determined by using various experimental techniques such as immunohistochemistry (IHC), Western blotting, fluorescence quantitative PCR (qPCR) and gene chips. Generally speaking, for a certain tumor antigen, its expression level is above a certain threshold and can be considered to be high expression. The specific threshold may vary depending on factors such as the purpose of the study, experimental technology and research background. In clinical studies, researchers usually determine the threshold based on the analysis of large sample data to determine whether the expression level of the tumor antigen is higher than the normal level. It should be noted that the high expression of tumor antigens may vary in different cancer types and may also vary in different patients.

[0285] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) form antibody binding sites respectively. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different types, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0286] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefrance et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0287] The CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably identified by the Kabat, Chothia or IMGT numbering systems.

[0288] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0289] As used herein, the term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means one of the two Fab' fragments formed by reducing the F(ab')2 fragment to break the disulfide bond at the F(ab')2 hinge region.

[0290] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 49) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 50) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol.

[0291] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody, which is a small antibody fragment with two antigen-binding sites, comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL) connected thereto in the same polypeptide chain (VH-VL or VL-VH), in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak RJ et al., Structure 2:1121 1123 (1994)).

[0292] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., the monoclonal antibodies ch517, ch704, and Hu517 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibodies can be screened for specificity in the same manner as for intact antibodies.

[0293] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0294] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and generally contain at least two or more different antibodies, which generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, USP 4,816,567).

[0295] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with an immunogen (and, if necessary, an adjuvant). The immunogen or adjuvant is typically injected subcutaneously at multiple sites or intraperitoneally. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM. After immunization, the animal will produce lymphocytes that secrete antibodies that specifically bind to the immunogen. Alternatively, lymphocytes can be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated and grown in a suitable culture medium, which preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells. For example, for parent myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of substances such as hypoxanthine, aminopterin, and thymidine to the culture medium (HAT medium) can inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA). There are also reports on the production of human monoclonal antibodies using human myeloma and human-mouse heteromyeloma cell lines (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined using the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107:220 (1980). After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned using the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture media may be DMEM or RPMI-1640. In addition, hybridoma cells can be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subcloned cells can be separated from the cell culture medium, ascites, or serum using traditional immunoglobulin purification methods, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0296] Monoclonal antibodies can also be obtained through genetic engineering and recombinant technology. Using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody, PCR amplification can be performed to isolate DNA molecules encoding the heavy and light chain genes of the monoclonal antibody from hybridoma cells. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0297] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).

[0298] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

[0299] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0300] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or different antigens. The multispecific antibody can be a full-length antibody or a fragment of the antibody. The term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens or that can recognize two different epitopes on a single antigen.

[0301] As used herein, the term "human antibody" or "fully human antibody" includes antibodies with variable regions and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies (i.e., humanized antibodies) in which CDR sequences derived from another mammalian species germline (such as mice) are grafted onto human framework sequences. Fully human antibodies or human antibodies can be derived from transgenic mice carrying human antibody genes or from human cells.

[0302] As used herein, the terms "specific binding," "specificity," or "specific for" refer to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0303] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., monoclonal antibodies ch517, ch704, hu517 of the present disclosure) have a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen (e.g., receptor tyrosine kinase-like orphan receptor 1 (ROR1)), e.g., as determined using surface plasmon resonance (SPR) in a BIACORE instrument.

[0304] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0305] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0306] As used herein, the term "polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a polypeptide may contain modifications, such as, but not limited to, optionally substituted polysarcosine residues, optionally substituted glycosylated polyethylene glycol, glycosylation, phosphorylation, or disulfide bond modifications. A "protein" may comprise one or more polypeptides.

[0307] As used herein, the terms "anti-ROR1 antibody," "anti-ROR1," "ROR1 antibody," "ROR1 monoclonal antibody," "antibody that binds to ROR1," and "antibody that targets ROR1" have the same meaning and refer to an antibody that can bind to ROR1 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic agent and / or therapeutic agent targeting ROR1.

[0308] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule such as amino acids or carbohydrates or sugar side chains and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0309] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

[0310] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0311] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0312] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the essential properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions in which residues are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0313] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, pH adjusters, surfactants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0314] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, inhibit, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered treatments, among others.

[0315] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor or infection) in a subject or to minimize its effect. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in the rate of disease progression, an improvement or alleviation of the disease state, and a regression or improved prognosis, whether detectable or undetectable. The amount of therapeutic agent that effectively alleviates any particular disease symptom can vary according to factors such as the patient's disease state, age, and weight, and the ability of the drug to elicit a desired response in the subject. Whether the disease symptoms are alleviated can be assessed by any clinical measurement, which is typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptoms.

[0316] As used herein, the terms "patient," "subject," "individual," and "subject" refer to any human or non-human animal, particularly a human, that is receiving prophylactic or therapeutic treatment. For example, the antibodies, methods, and compositions described herein can be used to treat a subject suffering from cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, horses, cats, chickens, mice, rats, amphibians, reptiles, and the like.

[0317] As used herein, the term "IC 50 "Half maximal inhibitory concentration" refers to the half-inhibitory concentration of the antagonist being measured. It indicates the half-dose of a drug or substance (inhibitor) that inhibits a biological process (or a substance involved in this process, such as an enzyme, cell receptor, or microorganism). 50 Can measure antibody sensitivity, IC 50 The lower the value, the higher the sensitivity of the antibody.

[0318] As used herein, the term "EC 50 "(concentration for 50% of maximal effect)" refers to the concentration that can cause 50% of the maximum effect.

[0319] As used herein, the term "antibody-drug conjugate (ADC)" is also referred to as antibody-drug conjugate, antibody-drug conjugate, or antibody-drug conjugate. Antibody-drug conjugates are composed of a monoclonal antibody targeting a specific antigen and a small molecule cytotoxic drug connected via a linker. They combine the powerful killing effect of traditional small molecule chemotherapy with the tumor targeting properties of antibody drugs. ADCs consist of three main parts: an antibody responsible for selectively recognizing antigens on the surface of cancer cells, a drug payload responsible for killing cancer cells, and a linker connecting the antibody and payload.

[0320] Beneficial technical effects

[0321] Experiments have shown that the main advantages of the present disclosure lie in one or more of the following aspects:

[0322] 1) mAb517 and mAb704 obtained in the present disclosure have better affinity for human ROR1 than the clinical antibody UC-961. ch517, ch704, and Hu517 obtained based on mAb517 and mAb704 can achieve better efficacy when coupled with equivalent drugs; and mAb517 and mAb704 obtained in the present disclosure can specifically bind to human ROR1. ch517, ch704, and Hu517 obtained based on mAb517 and mAb704 can also specifically bind to human ROR1.

[0323] 2) The antibody-drug conjugates obtained in the present disclosure, especially the antibody-drug conjugates comprising ch517 and its humanized Hu517, have better tumor inhibition effects than the control drugs in multiple tumor models; and the antibody-drug conjugates obtained in the present disclosure, especially the antibody-drug conjugates comprising Hu517, have significant bystander effects.

[0324] 3) The antibody-drug conjugates obtained in the present disclosure have good homogeneity and stability, especially the antibody-drug conjugates containing humanized Hu517, which have high in vivo stability and good pharmacokinetic properties, including a higher AUC and a longer half-life. BRIEF DESCRIPTION OF THE DRAWINGS

[0325] The accompanying drawings are used to provide an understanding of the technical solution of the present disclosure and constitute a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the technical solution of the present disclosure and do not constitute a limitation to the technical solution of the present disclosure.

[0326] Figure 1 shows representative binding curves of mAb517, mAb704, and UC-961 antibody (positive control antibody) binding to human ROR1;

[0327] Figure 2 shows the results of mAb517 and mAb704 antibodies binding to human ROR1 and human ROR2 as well as blank control plasmid transfected cells;

[0328] FIG3 shows a size exclusion chromatography (SEC) chromatogram of naked anti-ch517 prepared in Example 2 of the present disclosure;

[0329] Figure 4 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate ch517-T1000-exatecan prepared in Example 5 of the present disclosure;

[0330] Figure 5 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate ch517-T1000-exatecan prepared in Example 5 of the present disclosure;

[0331] FIG6 shows a size exclusion chromatography (SEC) chromatogram of naked antibody ch704 prepared in Example 2 of the present disclosure;

[0332] FIG7 shows a hydrophobic chromatography (HIC) chromatogram of naked antibody ch704 prepared in Example 2 of the present disclosure;

[0333] FIG8 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate ch704-T1000-exatecan prepared in Example 5 of the present disclosure;

[0334] Figure 9 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate ch704-T1000-exatecan prepared in Example 5 of the present disclosure;

[0335] FIG10 shows a size exclusion chromatography (SEC) chromatogram of naked antibody Hu517 prepared in Example 2 of the present disclosure;

[0336] FIG11 shows a hydrophobic chromatography (HIC) chromatogram of naked antibody Hu517 prepared in Example 2 of the present disclosure;

[0337] FIG12 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 of the present disclosure;

[0338] Figure 13 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 of the present disclosure;

[0339] FIG14 shows a size exclusion chromatography (SEC) chromatogram of naked antibody UC-961 prepared in the present disclosure;

[0340] FIG15 shows a hydrophobic chromatography (HIC) chromatogram of naked anti-UC-961 prepared in the present disclosure;

[0341] Figure 16 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate UC-961-T1000-exatecan prepared in Example 5 of the present disclosure;

[0342] Figure 17 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate UC-961-T1000-exatecan prepared in Example 5 of the present disclosure;

[0343] Figure 18 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate UC-961-vc-MMAE prepared in Example 5 of the present disclosure;

[0344] Figure 19 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate UC-961-vc-MMAE prepared in Example 5 of the present disclosure;

[0345] Figure 20 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 of the present disclosure;

[0346] Figure 21 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 of the present disclosure;

[0347] Figure 22 shows the size exclusion (SEC) chromatogram of the antibody-drug conjugate HuIgG-vc-MMAE prepared in Example 5 of the present disclosure;

[0348] Figure 23 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate HuIgG-vc-MMAE prepared in Example 5 of the present disclosure;

[0349] FIG24 shows a blood concentration-time curve of the antibody-drug conjugate Hu517-T1000-exatecan disclosed in the present invention;

[0350] Figure 25 shows a bar graph of the number of viable cells in a mixed cell line;

[0351] FIG26 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates in the Fadu mouse model;

[0352] FIG27 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human pancreatic cancer BXPC-3;

[0353] FIG28 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human lung adenocarcinoma NCI-H1975;

[0354] FIG29 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against triple-negative breast cancer MDA-MB-231;

[0355] FIG30 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human lung squamous cell carcinoma NCI-H226;

[0356] FIG31 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human non-small cell lung cancer PDX LD1-0025-200662;

[0357] FIG32 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human non-small cell lung cancer PDX LD1-0025-200730;

[0358] FIG33 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human triple-negative breast cancer PDX LD1-2009-362263.

[0359] Sequence information

[0360] The information of the partial sequences involved in the present disclosure is shown in Table 1 below.

[0361] Table 1. Information of partial sequences

[0362] The CDR region sequences in Table 1 above are defined using the Chothia, Kabat and IMGT numbering systems, respectively. Any other CDR region sequence determination method known in the art may also be used to identify the amino acid residues in the CDR region within the variable region. DETAILED DESCRIPTION

[0363] Example 1 Synthesis of Toxin Linkers

[0364] 1) Synthesis of compound LD-6

[0365] 1. Synthesis of intermediate LD6-C

[0366] Intermediate LD6-A (Mc-Val-Ala-OH, purchased from Shanghai Haoyuan Chemical, 2.4 g, 6.29 mmol) and LD6-B (3.18 g, 6.29 mmol) were dissolved in a mixture of dichloromethane and methanol (v:v = 2:1, 90 ml). p-EEDQ (2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 1.86 g, 7.55 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 24 hours and then evaporated to dryness under reduced pressure. LD6-C (3.9 g, 71% yield) was obtained by column chromatography. LC-MS (ESI, m / z) Calcd: 867.46, Found: 868.49 (M+H).

[0367] 2. Synthesis of intermediate LD6-D

[0368] Intermediate LD6-C (2 g, 2.3 mmol) was dissolved in 50 ml of anhydrous tetrahydrofuran and cooled in an ice bath under argon. Pyridine hydrofluoride complex (4.6 g, 46 mmol) was added and the reaction mixture was stirred at 0°C for 2 hours. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was separated, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain intermediate LD6-D (1.1 g, 76% yield). LC-MS (ESI, m / z) Required: 629.34, Found: 630.31 (M+H).

[0369] 3. Synthesis of intermediate LD6-E

[0370] Intermediate LD6-D (700 mg, 1.11 mmol) was dissolved in 4 ml of anhydrous DMF. DIPEA (0.39 ml, 2.23 mmol) and bis(4-nitrophenyl) carbonate (406 mg, 1.33 mmol) were added at room temperature. The mixture was stirred overnight at room temperature under argon. The solvent was evaporated under reduced pressure, and methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered and rinsed several times with diethyl ether. After drying, the solid obtained intermediate LD6-E, which was used directly in the next reaction. LC-MS (ESI, m / z) Required: 794.35, Found: 795.41 (M+H).

[0371] 4. Synthesis of intermediate LD6-F

[0372] Intermediate LD6-E (300 mg, 0.44 mmol) was dissolved in 400 μL of anhydrous DMF, and 100 μL of anhydrous pyridine was added. Exatecan mesylate (purchased from Shanghai Haoyuan, 234 mg, 0.44 mmol) and HOBt (60 mg, 0.44 mmol) were then added. The mixture was stirred at room temperature overnight under argon. The reaction mixture was purified by reverse-phase HPLC to obtain intermediate LD6-F (230 mg, 48% yield). LC-MS (ESI, m / z) Required: 1090.48, Found: 1091.53 (M+H).

[0373] 5. Synthesis of intermediate LD6-G

[0374] Intermediate LD6-F (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous dichloromethane. 300 μL of trifluoroacetic acid was added under ice-cooling. The mixture was returned to room temperature and stirred for 30 minutes. The solvent was removed under reduced pressure to obtain the final product, the trifluoroacetate salt of intermediate LD6-G, which was used directly in the next reaction without further purification. LC-MS (ESI, m / z) Required: 990.43, Found: 991.47 (M+H).

[0375] 6. Synthesis of compound (LD-6)

[0376] Compound LD6-G (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF. Acetyl-10-polysarcosine (Ac-Sar10-COOH, 84 mg, 0.109 mmol), HATU (50 mg, 0.130 mmol), and DIPEA (38 μL, 0.22 mmol) were added, respectively. The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure to obtain the final product, which was then purified by reverse-phase HPLC to obtain compound (LD-6) (74 mg, 38% yield). LC-MS (ESI, m / z) Required: 1742.81, Found: 1743.85 (M+H).

[0377] 2) Synthesis of compound LD-9

[0378] The synthesis of compound LD-9 followed the same steps and conditions as compound LD-6, except that the starting material in the final step, acetylated-10 polysarcosine, was replaced with GPEG-1 (synthesized according to the methods described in CN107109405A and CN110214141A). LC-MS (ESI, m / z) Calcd: 1903.90, Found: 1904.73 (M+H).

[0379] 4) Synthesis of compounds LD-3, LD-4, LD-5, LD-7, LD-8, LD-10 and LD-11

[0380] Referring to the synthesis method of compound LD-6, compounds LD-3, LD-4, LD-5, LD-7, LD-8, LD-10 and LD-11 were synthesized. Their structural formulas are shown in Table 2 below. The relevant intermediates for preparing these compounds can be purchased commercially.

[0381] Table 2

[0382] Example 2 Preparation of Antibodies

[0383] ROR1 monoclonal antibody preparation

[0384] Balb / c mice (8-12 weeks old) were immunized with a human ROR1 protein extracellular domain fragment antigen (NCBI reference sequence: NP_005003.2, amino acid sequence positions 30-403), and their serum titers were monitored to determine the number of immunizations. After the initial immunization, 3 to 4 booster immunizations were performed, and the mouse serum was taken to detect the titer according to conventional methods in the art. After the mice that passed the titer test were immunized once, the whole spleen and 1 / 2 of the lymph nodes were taken and PEG fused with the myeloma SP2 / 0 cell line. The fused cells were plated and cultured, and the supernatants of all wells were collected. The antibodies were screened using the ELISA method, and the positive wells with cells under the microscope were transferred to 96-well plates for further culture. After 7 days of growth, the supernatants of all wells were collected and the reaction with the antigen was detected using the ELISA method. The positive wells were further tested for antigen binding at different dilutions to perform affinity sorting. The 20 parent clones with the highest immunogen affinity were subcloned. Subcloning was performed by limiting dilution and ELISA screening to obtain two hybridoma cell lines. Mouse ascites was prepared from the two hybridoma cell lines, and two mouse monoclonal antibodies (abbreviated as "mAb517" and "mAb704") were obtained after collection and purification.

[0385] The sequence information of the mouse ROR1 monoclonal antibody (mAb517) is as follows:

[0386] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 1-3; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 4-6, or

[0387] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 7-9; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 10-12, or

[0388] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 13-15; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 16-18.

[0389] The sequence information of the mouse ROR1 monoclonal antibody (mAb704) is as follows:

[0390] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 19-21; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 22-24, or

[0391] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 25-27; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 28-30, or

[0392] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 31-33; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 34-36. Based on the CDRs of the murine ROR1 monoclonal antibody, the hybridoma sequences were humanized to generate chimeric ROR1 antibodies (abbreviated as "ch517" and "ch704") and a humanized ROR1 antibody (abbreviated as "Hu517"), the sequences of which are shown in Table 3.

[0393] Table 3. Amino acid sequences of the heavy and light chain variable regions of chimeric and humanized ROR1 antibodies

[0394] The sequence information of the humanized ROR1 antibody (Hu517) is as follows:

[0395] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 1, 45, and 3; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 46, 5, and 6, or

[0396] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 7-9; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 47, 11, 12, or

[0397] The amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 13-15; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 16-18.

[0398] Preparation of human-mouse chimeric antibodies

[0399] The ch517 heavy chain and light chain variable region sequences (see SEQ ID NO: 37, SEQ ID NO: 38) and the ch704 heavy chain and light chain variable region sequences (see SEQ ID NO: 39, SEQ ID NO: 40) were cloned into a vector containing the human IgG1 heavy chain constant region and the kappa chain constant region by gene recombination technology. After sequencing, the vector was transfected and expressed in a mammalian expression system (FreeStyle TM The constructed chimeric antibodies were expressed in 293T cells and then purified. The obtained human-mouse chimeric antibodies were named ch517 and ch704, and the sequences of their heavy and light chain variable regions are shown in Table 1. The positive control antibody UC-961 was prepared using the same method. Its antibody sequence is shown in SEQ ID NO. 2 and SEQ ID NO. 4 in patent WO2021159029 A1. The full-length sequences of the heavy and light chains of UC-961 (the full-length sequences described in Ab1 light chain and Ab1 heavy chain in WO2018237335A1) were used to construct pCDNA3.1 expression vectors, respectively. The antibodies were expressed in 293F suspension cell culture and purified by protein A / G gravity column.

[0400] Preparation of humanized antibodies

[0401] The humanized template that best matched the non-CDR region of ch517 was searched and selected from the Germline database. The CDR region of the antibody was then transplanted onto the selected humanized template to replace the CDR region of the human template, and then recombined with the IgG1 constant region. At the same time, based on the three-dimensional structure of the mouse antibody, back mutations were performed on the embedded residues, residues that directly interacted with the CDR region, and residues that had an important impact on the conformation of VL and VH.

[0402] The designed humanized variable region sequence was cloned into a vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region by gene recombination technology. After sequencing, the humanized variable region sequence was expressed using transfection technology and mammalian expression system (FreeStyle TM The constructed humanized antibody was expressed in 293 cells and then purified to obtain a humanized antibody Hu517. The sequences of its heavy chain and light chain variable regions are shown in Table 1.

[0403] Example 3 Affinity detection of anti-ROR1 antibodies in human ROR1 recombinant protein ELISA

[0404] Human ROR1 protein (catalog number: RO1-H522y, Acrobiosystems) was fixed to a 96-well plate by incubation overnight at 4°C. The plate was then blocked by incubation at 37°C with 1% BSA in PBS for 1 hour. After blocking, the plate was washed 3 times with PBST (PBS containing 0.05% Tween20). Serial dilutions of anti-ROR1 antibodies were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the fixed protein at 37°C for 1 hour. After binding, the plate was washed 3 times with PBST, incubated at 37°C with peroxidase-labeled goat anti-mouse IgG or goat anti-human IgG (Jackson Immuno Research) diluted 1 / 10,000 in binding buffer for 1 hour, washed again, developed with TMB and terminated with 1M H2SO4. The absorbance at 450nm-620nm was measured.

[0405] Representative binding curves of mAb517, mAb704, and UC-961 (positive control antibody) for binding to human ROR1 are shown in Figure 1, and the EC50s are shown in Table 4. The results show that all three antibodies specifically bind to human ROR1 protein, and the affinity of mAb517 and mAb704 antibodies is significantly better than that of UC-961 (positive control antibody).

[0406] Table 4. EC50 of mAb517, mAb704 and UC-961 antibodies binding to human ROR1

[0407] Example 4: Determining the Binding Specificity of Antibodies to Human ROR1 and Human ROR2 Using Overexpression Plasmid Transfection Flow Cytometry

[0408] 293T cells (human embryonic kidney cells, cell bank of the Chinese Academy of Sciences, catalog number: SCSP-502) were cultured to a cell density of 80%. Cells were collected and plated on 6-well plates. The cell density was adjusted to 2-5 × 10 5 / ml. 3mL was plated in each well. On the second day, plasmid transfection was performed. 3μg of human ROR1 plasmid and 3μg of human ROR2 plasmid were mixed with 9μL PEI respectively and allowed to stand for 20 minutes. During this period, serum-free medium was replaced. The plasmid after standing was added to the cells, mixed well, and returned to the incubator. After 6 hours, complete medium was replaced and cultured overnight. On the third day, the cells were removed and digested with trypsin. The digested cells were centrifuged at 1000rpm for 5 minutes and then blocked with 10% goat serum (purchased from Beijing Solebow Technology Co., Ltd., catalog number SL038) for 30 minutes. Anti-ROR1 antibodies mAb517 and mAb704 were added to the cells at 100nM and incubated for 1 hour. After washing with PBS twice, APC-labeled goat anti-mouse secondary antibody (purchased from Jackson ImmunoResarch, diluted 1:500) was added and incubated for 1 hour. Finally, the results were analyzed by flow cytometry after washing with PBS twice.

[0409] The results of mAb517 and mAb704 binding to human ROR1 and human ROR2, as well as blank control plasmid-transfected cells, are shown in Figure 2. The results show that both mAb517 and mAb704 specifically bind to human ROR1-transfected cells but not to human ROR2-transfected cells. Furthermore, mAb517 and mAb704 showed no binding reaction in blank control plasmid-transfected cells.

[0410] Example 5 Preparation of Antibody Drug Conjugates

[0411] 1) General operation method

[0412] The prepared antibody-drug conjugate (Formula (I)) can be concentrated, liquid-changed, purified, and the antibody concentration measured and the average number of drug molecules carried by each antibody calculated according to the common operations described below to identify the antibody-drug conjugate.

[0413] Procedure A: Concentration of Antibodies or Antibody-Drug Conjugates

[0414] Take an ultrafiltration tube (Amicon Ultra, 50000 MWCO, Millipore Corporation), add the antibody or antibody-drug conjugate solution to be concentrated, centrifuge to the required volume and then take out.

[0415] Procedure B: Measurement of Antibody Concentration

[0416] The absorption of the antibody was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) according to the manufacturer's method. The ratio of the absorption to the absorption coefficient of the antibody at that wavelength was the concentration of the antibody. Here, the characteristic absorption wavelength of the antibody was 280 nm, and the absorption coefficient of the antibody at this wavelength is usually 1.3 mL mg -1 cm-1 to 1.8mL mg -1 cm -1 between.

[0417] Procedure C: Antibody exchange

[0418] A Zeba desalting column (5 mL, 40K MWCO) was pre-equilibrated with phosphate buffer (50 mM, pH 7.0) containing sodium chloride (50 mM) and EDTA (2 mM) according to the manufacturer's instructions (Thermo Fisher Scientific). 2 mL of sample was loaded onto each Zeba desalting column (5 mL), centrifuged (1000 g, 4 min), and the flow-through fraction collected. This fraction was concentrated using Procedure A and the antibody concentration was determined using Procedure B, adjusting the antibody concentration using PBS 7.0 / EDTA.

[0419] Procedure D: Purification of Antibody-Drug Conjugates

[0420] According to the manufacturer's instructions (Thermo Fisher Scientific), a Zeba desalting column (5 mL, 40K MWCO) was pre-equilibrated with any suitable storage buffer. Storage buffers include, but are not limited to, histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM sodium chloride and phosphate buffer (50 mM, pH 7.0) containing 50 mM sodium chloride. The reaction solution containing the antibody-drug conjugate (approximately 2 mL) was added to a 5 mL Zeba desalting column and centrifuged (1000 g, 4 minutes). The flow-through fraction (approximately 2 mL) was collected. This method utilizes a gel filtration purification process. The collected fractions were applied to the desalting column and centrifuged and eluted. The elution process was repeated twice to remove unbound drug linkers and low molecular weight compounds such as the reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-(L)-cysteine ​​(NAC), and dimethyl sulfoxide to obtain the antibody-drug conjugate.

[0421] Procedure E: Measure the concentration of the antibody in the antibody-drug conjugate and the average number of drug molecules attached to each antibody (DAR value)

[0422] The concentration of the drug coupled to the antibody-drug conjugate can be obtained by measuring the ultraviolet absorbance of the antibody-drug conjugate aqueous solution at 280 nm and 370 nm and calculating using the following formula.

[0423] At any given wavelength, the total absorbance of a system is equal to the sum of the absorbances of all light-absorbing chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar absorption coefficients of the antibody and drug remain unchanged before and after conjugation, the antibody concentration and drug concentration in the antibody-drug conjugate can be expressed by the following formulas: A280 = AD,280 + AA,280 = εD,280CD + εA,280CA Formula (1) A370 = AD,370 + AA,370 = εD,370CD + εA,370CA Formula (2)

[0424] Here, A280 represents the total absorbance of the antibody-drug conjugate aqueous solution at 280 nm, A370 represents the total absorbance of the antibody-drug conjugate aqueous solution at 370 nm, AA,280 represents the absorbance of the antibody at 280 nm, AA,370 represents the absorbance of the antibody at 370 nm, AD,280 represents the absorbance of the drug molecule at 280 nm, AD,370 represents the absorbance of the drug molecule at 370 nm, εA,280 represents the molar extinction coefficient of the antibody at 280 nm, εA,370 represents the molar extinction coefficient of the antibody at 370 nm, εD,280 represents the molar extinction coefficient of the drug molecule at 280 nm, εD,370 represents the molar extinction coefficient of the drug molecule at 370 nm, CA represents the antibody concentration in the antibody-drug conjugate, and CD represents the drug concentration in the antibody-drug conjugate.

[0425] In this case, εA,280, εA,370, εD,280, and εD,370 are all known values ​​(calculated from the antibody sequence or measured by UV absorbance of the compound). For example, εA,280 can be calculated from the amino acid sequence of the antibody using a known method (Protein Science, 1995, Vol. 4, pp. 2411-2423). Antibodies typically do not absorb at 370 nm, so εA,370 is typically 0. εD,280 and εD,370 can be calculated by measuring the absorbance of the drug molecule at 280 nm and 370 nm as a function of concentration and then using the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × pathlength). CA and CD can be obtained by measuring the absorbance values ​​of the antibody-drug conjugate at 280 nm and 370 nm, respectively, and then solving the two-variable linear equation system (Equations (1) and (2)). In addition, the average number of drug molecules attached to each antibody (DAR value) can be obtained by dividing CD by CA.

[0426] Procedure F: Measurement of Aggregates in Antibody-Drug Conjugates

[0427] Aggregates in antibody-drug conjugates can be measured by size exclusion chromatography in high performance liquid chromatography. The analytical method is as follows:

[0428] High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0429] Detector: Ultraviolet absorption spectrometer (detection wavelength: 280nm)

[0430] Column model: TOSOH TSKgel G3000SWXL (7.8×300mm, 5μm)

[0431] Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0

[0432] Flow rate: 0.75 mL / min

[0433] Analysis time: 18 minutes

[0434] Column temperature: room temperature

[0435] Injection volume: 50 μg

[0436] Data Analysis: The size exclusion chromatogram of the quality control (QC, Hu517 naked antibody) is shown in Figure 10. Notably, under this analytical method, the main peak (single peak) of the 150 kDa QC has a retention time between 9.5 and 10.5 minutes. Furthermore, the retention time of aggregates should be earlier than that of the monomers.

[0437] Procedure G: Comparison of hydrophobicity of antibody-drug conjugates and determination of DAR values

[0438] The hydrophobicity of antibody-drug conjugates can be analyzed on a high performance liquid chromatography (HPLC) column using a hydrophobic column. The analysis method is as follows:

[0439] High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0440] Detector: Ultraviolet absorption spectrometer (detection wavelength: 280nm)

[0441] Column model: TOSOH TSKgel Butyl-NPR (4.6mm ID×3.5cm, 2.5μm)

[0442] Mobile phase A: mol / L (NH4)2SO4, 50mmol / L KHPO4, pH 7.0

[0443] Mobile phase B: 50 mmol / L KHPO4, 25% (v / v) isopropanol, pH 7.0

[0444] Analysis time: 25 minutes

[0445] Column temperature: room temperature

[0446] Elution program (B%): 0%-25% (0-1 min), 25% (1-3 min), 25%-80% (3-13 min), 80% (13-17 min), 80%-0% (17-17.10 min), 0% (17.10-25 min)

[0447] Injection volume: 10 μL

[0448] Data Analysis: The hydrophobicity chromatography chromatogram of the quality control (QC, naked Hu517 antibody) is shown in Figure 11. Compared to naked antibodies without drug molecules, antibody-drug conjugates have higher hydrophobicity, resulting in longer retention times. It is well known that shorter retention times indicate weaker hydrophobicity. The DAR value of the antibody-drug conjugate can be calculated.

[0449] II) Preparation of antibody-drug conjugate ch517-T1000-exatecan

[0450] Step: Antibody-Drug Conjugate

[0451] Antibody reduction: Follow the above procedure B (the extinction coefficient of the antibody at 280 nm is 1.462 mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 8.6 mg / mL. Add 0.184 mL of 5 mM TCEP solution (equivalent to 7 equivalents of the antibody content) to 2.3 mL of the ch517 protein aqueous solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0452] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 0.66 mL of the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 25 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0453] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate ch517-T1000-exatecan.

[0454] Characterization of Antibody-Drug Conjugates: Using the above procedure E(ε D,280 =6384 and ε D,370 =16180), and the obtained antibody-drug conjugate was characterized by operation F and operation G.

[0455] The concentration of the antibody-drug conjugate was calculated to be 7.85 mg / mL using procedure E. The average number of small molecules conjugated per antibody was calculated to be 8.86, resulting in a DAR value of approximately 8.86. The aggregate content of naked anti-ch517 was measured to be 2.55% (Figure 3), and the aggregate content of the antibody-drug conjugate ch517-T1000-exatecan was 6.58% (Figure 4). The retention time of the antibody-drug conjugate ch517-T1000-exatecan was measured to be 6.718 min using procedure G (Figure 5).

[0456] III) Preparation of antibody-drug conjugate ch704-T1000-exatecan

[0457] Step: Antibody-Drug Conjugate

[0458] Antibody reduction: Follow the above procedure B (antibody extinction coefficient at 280 nm is 1.48 mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 7.2 mg / mL. Add 0.184 mL of 5 mM TCEP solution (equivalent to 7 equivalents of the antibody content) to 2.75 mL of the ch704 protein aqueous solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0459] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 0.66 mL of the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 25 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0460] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate ch704-T1000-exatecan.

[0461] Characterization of Antibody-Drug Conjugates: Using the above procedure E(ε D,280 =6384 and ε D,370 =16180), and the obtained antibody-drug conjugate was characterized by operation F and operation G.

[0462] The concentration of the antibody-drug conjugate was calculated to be 8.01 mg / mL using procedure E. The average number of small molecules conjugated per antibody was calculated to be 8.68, resulting in a DAR value of approximately 8.68. The aggregate content of naked anti-ch704 was measured to be 13.21% ( FIG. 6 ), while the aggregate content of the antibody-drug conjugate ch704-T1000-exatecan was 1.54% ( FIG. 8 ). The retention time of naked anti-ch704 was measured to be 3.527 min ( FIG. 7 ), while the retention time of the antibody-drug conjugate ch704-T1000-exatecan was 6.423 min ( FIG. 9 ), using procedure G.

[0463] IV) Preparation of Antibody-Drug Conjugate Hu517-T1000-exatecan

[0464] Step: Antibody-Drug Conjugate

[0465] Antibody reduction: Follow the above procedure B (the extinction coefficient of the antibody at 280 nm is 1.444 mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 29.47 mg / mL. Add 0.307 mL of 5 mM TCEP solution (equivalent to 9 equivalents of the antibody content) to 0.87 mL of the Hu517 protein aqueous solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0466] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 0.24 mL of the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 14 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0467] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate Hu517-T1000-exatecan.

[0468] Characterization of Antibody-Drug Conjugates: Using the above procedure E(ε D,280 =6384 and ε D,370 =16180), and the obtained antibody-drug conjugate was characterized by operation F and operation G.

[0469] The concentration of the antibody-drug conjugate was calculated to be 7.21 mg / mL by operation E, and the average number of small molecules coupled per antibody was calculated to be 7.91 by operation E, i.e., the DAR value was approximately 7.91. The aggregate content of naked anti-Hu517 measured by operation F was 0.26% (Figure 10), and the aggregate content of the antibody-drug conjugate Hu517-T1000-exatecan was 1.08% (Figure 12). The retention time of naked anti-Hu517 measured by operation G was 3.707 min (Figure 11), and the retention time of the antibody-drug conjugate Hu517-T1000-exatecan was 6.653 min (Figure 13).

[0470] V) Preparation of Antibody-Drug Conjugate UC-961-T1000-exatecan

[0471] Step: Antibody-Drug Conjugate

[0472] Antibody reduction: Follow the above procedure B (the extinction coefficient of the antibody at 280 nm is 1.505 mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 6.71 mg / mL. Add 0.24 mL of a 5 mM TCEP solution (equivalent to 9 equivalents of the antibody content) to 2.98 mL of the UC-961 protein aqueous solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0473] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 0.186 mL of the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 14 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0474] Purification of the antibody-drug conjugate: The reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate UC-961-T1000-exatecan.

[0475] Characterization of Antibody-Drug Conjugates: Using the above procedure E(ε D,280 =6384 and ε D,370 =16180), and the obtained antibody-drug conjugate was characterized by operation F and operation G.

[0476] The concentration of the antibody-drug conjugate was calculated to be 5.46 mg / mL by operation E, and the average number of small molecules coupled to each antibody was calculated to be 8.62 by operation E, i.e., the DAR value was approximately 8.62. The aggregate content of naked anti-UC-961 was measured to be 7.15% (Figure 14), and the aggregate content of the antibody-drug conjugate UC-961-T1000-exatecan was 3.01% (Figure 16). The retention time of naked anti-UC-961 measured by operation G was 6.293 min (Figure 15), and the retention time of the antibody-drug conjugate UC-961-T1000-exatecan was 7.534 min (Figure 17).

[0477] VI) Preparation of Antibody-Drug Conjugate UC-961-vc-MMAE

[0478] Step: Antibody-Drug Conjugate

[0479] Antibody reduction: Follow the above procedure B (the extinction coefficient of the antibody at 280 nm is 1.505 mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA. The antibody concentration after replacement is 7.92 mg / mL. Add 61.87 μL of 5 mM TCEP solution (equivalent to 3.2 equivalents of the antibody content) to 1.83 mL of the UC-961 protein aqueous solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0480] Conjugation between the antibody and the drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 135.33 μL of the drug linker vc-MMAE (purchased from Shanghai Haoyuan Chemical, catalog number: HY-15575) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 7 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0481] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate UC-961-vc-MMAE.

[0482] Characterization of Antibody-Drug Conjugates: The obtained antibody-drug conjugates were characterized using the above-mentioned procedures E, F, and G.

[0483] The concentration of the antibody-drug conjugate was calculated to be 4.08 mg / mL by operation E. The aggregate content of the antibody-drug conjugate UC-961-vc-MMAE was measured to be 2.53% by operation F ( FIG. 18 ). FIG. 19 is a hydrophobic chromatography profile of the antibody-drug conjugate UC-961-vc-MMAE measured by operation G. The average number of drug molecules attached to each antibody was calculated to be 4.49, i.e., the DAR value was approximately 4.49 ( FIG. 19 ).

[0484] VII) Preparation of Antibody-Drug Conjugate HuIgG-T1000-exatecan

[0485] Step: Antibody-Drug Conjugate

[0486] Antibody reduction: Follow the above procedure B (antibody extinction coefficient at 280nm is 1.35mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 15 mg / mL. Add 0.27 mL of 5 mM TCEP solution (equivalent to 10 equivalents of the antibody content) to 1.33 mL of HuIgG protein (Sino Biological, Cat: HG1K) in water, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0487] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 0.2 mL of the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) (equivalent to 15 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0488] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in Procedure D to obtain the antibody-drug conjugate HuIgG-T1000-exatecan.

[0489] Characterization of Antibody-Drug Conjugates: Using the above procedure E(ε D,280 =6384 and εD,370 =16180), and the obtained antibody-drug conjugate was characterized by operation F and operation G.

[0490] The concentration of the antibody-drug conjugate was calculated to be 12.09 mg / mL using procedure E. The average number of small molecules conjugated per antibody was calculated to be 6.71, i.e., the DAR value was approximately 6.71. The aggregate content of the antibody-drug conjugate HuIgG-T1000-exatecan was measured to be 4.38% using procedure F ( FIG. 20 ). The retention time of the antibody-drug conjugate HuIgG-T1000-exatecan, measured using procedure G, was 6.213 min ( FIG. 21 ).

[0491] VIII) Preparation of Antibody-Drug Conjugate HuIgG-vc-MMAE

[0492] Step: Antibody-Drug Conjugate

[0493] Antibody reduction: Follow the above procedure B (antibody extinction coefficient at 280nm is 1.35mL mg -1 cm -1 ) and Procedure C: Replace the antibody buffer with PBS 7.0 buffer containing EDTA to a concentration of 15 mg / mL. Add 40 μL of 5 mM TCEP solution (equivalent to 2.5 equivalents of the antibody content) to 0.8 mL of the HuIgG protein solution, ensuring the pH of the reaction solution is 7.0 ± 0.1. Incubate the mixture at 37°C for 2 hours.

[0494] Conjugation of the antibody and drug linker: Incubate the mixture at 4°C for 10 minutes. Next, add 112 μL of the drug linker vc-MMAE dissolved in N,N-dimethylacetamide (DMA) (equivalent to 7 times the antibody content). Incubate the mixture at 22°C for 30 minutes.

[0495] Purification of antibody-drug conjugate: The above reaction solution was purified using the method described in operation D to finally obtain the antibody-drug conjugate HuIgG-vc-MMAE.

[0496] Characterization of Antibody-Drug Conjugates: The obtained antibody-drug conjugates were characterized using the above-mentioned procedures E, F, and G.

[0497] The concentration of the antibody drug conjugate was calculated by operation E to be 9.21 mg / mL. The aggregate content of the antibody-drug conjugate HuIgG-vc-MMAE was measured by operation F to be 7.67% (Figure 22). Figure 23 is a hydrophobic chromatography profile of the antibody-drug conjugate HuIgG-vc-MMAE measured by operation G.

[0498] The structural formulas of the prepared antibody-drug conjugates ch517-T1000-exatecan, ch704-T1000-exatecan, Hu517-T1000-exatecan, UC-961-T1000-exatecan, and HuIgG-T1000-exatecan are shown in C-6, where Ab and m represent antibody and DAR value, respectively.

[0499] The structural formulas of the prepared antibody-drug conjugates UC-961-vc-MMAE and HuIgG-vc-MMAE are shown in C-11, wherein Ab and m represent the antibody and DAR value, respectively.

[0500] IX) Preparation of other antibody-drug conjugates

[0501] Antibody-drug conjugates C-3, C-4, C-5, C-7, C-8, C-9, C-10, and C-11 were prepared according to the general operating procedures. Their structural formulas are shown in the following table, where m represents the DAR value, which is about 8 or about 4; Ab represents the antibody, which is ch517, ch704, or Hu517.

[0502] Example 6 Pharmacokinetics of ROR1 Antibody-Drug Conjugate (Hu517-T1000-exatecan) in Nude Mice

[0503] Six-week-old female BALB / C nude mice, weighing 18-21 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 was administered intravenously at a dose of 10 mg / kg once weekly for a total of one dose. Blood was collected from the mice at 15 minutes, 4 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, and 21 days after administration. ELISA assays were performed to assess drug concentrations at different time points, and pharmacokinetic parameters were calculated. Figure 24 shows the plasma concentration-time curve of the antibody-drug conjugate Hu517-T1000-exatecan, and Table 5 shows the associated pharmacokinetic parameters.

[0504] Table 5. Pharmacokinetic parameters of Hu517-T1000-exatecan

[0505] The results showed that the ADC concentration and total antibody concentration curves of the drug had a good degree of overlap, proving that the drug was highly stable. At the same time, the pharmacokinetic parameters showed that the drug had a higher AUC parameter and a longer half-life.

[0506] Example 7 Evaluation of the in vitro bystander effect of ROR1 antibody-drug conjugate (Hu517-T1000-exatecan)

[0507] PC9 (human lung cancer cells, Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-5085) and A549 (human lung cancer cells, ATCC, catalog number: CCL-185) were cultured to a cell density of 80%. The cells were collected and mixed with PC9 (ROR1-positive cells) and A549 (ROR1-negative cells) at a ratio of 1:3 and plated on 6-well plates. The mixed cell density was adjusted to 2-5 × 10 4 / mL, 3mL was plated per well. The ADC molecule and negative control were added to the mixed cell culture medium at a working concentration of 100nM, respectively, and the cells were incubated for 5 days. Cell apoptosis was observed regularly during this period. After 5 days, the cells were removed, digested with trypsin, and counted. The total number of viable cells in the ADC molecule and negative control wells was calculated. The digested cells were centrifuged at 1000rpm for 5 minutes and blocked with 10% goat serum (purchased from Beijing Solebao Technology Co., Ltd., Cat. No. SL038) for 30 minutes. The anti-ROR1 humanized antibody Hu517 was added to the cells as a primary antibody at 100nM and incubated for 1 hour. After washing twice with PBS, FITC-conjugated goat anti-human secondary antibody (purchased from Jackson ImmunoResearch) was added at a dilution of 1:500 and incubated for 1 hour. Finally, the cells were washed twice with PBS and analyzed by flow cytometry. The number of viable PC9 and A549 cells was calculated based on the test results, and a bar graph of cell survival was plotted.

[0508] Figure 25 is a bar graph showing the number of viable cells in mixed cell lines containing either Hu517-T1000-exatecan or HuIgG-T1000-exatecan, the antibody-drug conjugates prepared in Example 5. The results demonstrate that, compared to the negative antibody-drug conjugate HuIgG-T1000-exatecan, Hu517-T1000-exatecan exhibited a stronger killing effect not only on ROR1-positive PC9 cells but also on ROR1-negative A549 cells, demonstrating a significant bystander effect.

[0509] Example 8 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human head and neck squamous cell carcinoma CDX Fadu

[0510] Six-week-old female BALB / C nude mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Five million Fadu cells (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu132) were inoculated subcutaneously in each mouse. 3 The antibody-drug conjugates ch517-T1000-exatecan and HuIgG-T1000-exatecan prepared in Example 5 were administered intravenously at a dose of 10 mg / kg; the antibody-drug conjugates UC-961-vc-MMAE and HuIgG-vc-MMAE prepared in Example 5 were administered intravenously at a dose of 5 mg / kg. Dosing was performed once weekly (QW) for a total of two doses. Tumor volume was measured twice weekly after dosing using a vernier caliper and calculated according to the following formula: TV = (length * width^2) / 2.

[0511] Figure 26 shows the in vivo tumor volume-time change curve of the Fadu mouse model. The results show that compared with the negative control, ch517-T1000-exatecan significantly inhibited tumor growth, while UC-961-vc-MMAE did not produce any tumor growth inhibitory effect.

[0512] Example 9 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human pancreatic cancer CDX BXPC-3

[0513] Six-week-old female BALB / C nude mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Five million BXPC-3 cells (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu 12) were subcutaneously inoculated into the mice. When the average tumor volume reached 150 mm 3 The antibody-drug conjugates ch517-T1000-exatecan and ch704-T1000-exatecan prepared in Example 5 were administered intravenously at a dose of 10 mg / kg. A negative control group, HuIgG-T1000-exatecan, was also administered intravenously. Dosing was performed once weekly (QW) for a total of three doses. Tumor volume was measured twice weekly using a vernier caliper after dosing and calculated using the following formula: TV = (length * width²) / 2.

[0514] Figure 27 shows the in vivo tumor volume-time change curve of the pancreatic cancer mouse model. The results show that compared with the negative control HuIgG-T1000-exatecan and PBS groups, both ch517-T1000-exatecan and ch704-T1000-exatecan can partially inhibit tumor growth, and the tumor inhibition trend and duration of ch517-T1000-exatecan are better than those of ch704-T1000-exatecan.

[0515] Example 10 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human lung adenocarcinoma CDX NCI-H1975

[0516] Six-week-old female BALB / C nude mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Five million NCI-H1975 cells (Cell Bank of the Chinese Academy of Sciences, Catalog No. SCSP-597) were subcutaneously inoculated into the mice. When the average tumor volume reached 150 mm 3 The antibody-drug conjugates ch517-T1000-exatecan and ch704-T1000-exatecan prepared in Example 5 were administered intravenously at a dose of 10 mg / kg. A negative control group, HuIgG-T1000-exatecan prepared in Example 5, was also administered intravenously with an equal dose of PBS. Dosing was performed once weekly for a total of one dose. Tumor volume was measured twice weekly using a vernier caliper after dosing and calculated using the following formula: TV = (length * width²) / 2.

[0517] Figure 28 shows the in vivo tumor volume-time change curve of the lung adenocarcinoma mouse model. The results show that compared with the negative control HuIgG-T1000-exatecan and PBS groups, both ch517-T1000-exatecan and ch704-T1000-exatecan can significantly inhibit tumor growth, and the tumor inhibition trend and duration of ch517-T1000-exatecan are better than those of ch704-T1000-exatecan.

[0518] Example 11 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human triple-negative breast cancer CDX MDA-MB-231

[0519] Six-week-old female BALB / C nude mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Five million MDA-MB-231 cells (Cell Bank of the Chinese Academy of Sciences, Catalog No. SCSP-5043) were subcutaneously inoculated into the mice. When the average tumor volume reached 150 mm 3 The antibody-drug conjugates Hu517-T1000-exatecan, UC-961-vc-MMAE, and UC-961-T1000-exatecan prepared in Example 5 were administered intravenously at a dose of 5 mg / kg. A negative control group, Vehicle, received an equal dose of PBS intravenously. Dosing was performed once weekly (QW) for a total of three doses, with the UC-961-vc-MMAE group receiving a total of five doses. Tumor volume was measured twice weekly using a vernier caliper after dosing, and calculated using the following formula: TV = (length * width²) / 2.

[0520] Figure 29 shows the in vivo tumor volume-time change curve of the triple-negative breast cancer mouse model. The results show that compared with the negative control, Hu517-T1000-exatecan, UC-961-T1000-exatecan and UC-961-vc-MMAE can all significantly inhibit tumor growth. The tumor inhibition trend of Hu517-T1000-exatecan is better than that of UC-961-T1000-exatecan, and UC-961-vx-MMAE has the weakest tumor inhibition.

[0521] Example 12 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human lung squamous cell carcinoma CDX NCI-H226

[0522] Six-week-old female NU / NU mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Five million NCI-H226 cells (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu235) were subcutaneously inoculated into the mice. When the average tumor volume reached 150 mm 3The antibody-drug conjugates Hu517-T1000-exatecan and UC-961-vc-MMAE prepared in Example 5 were administered intravenously at doses of 10 mg / kg and 5 mg / kg, respectively. The antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 was administered intravenously at a dose of 10 mg / kg. A negative control group, Vehicle, was also administered with an equal dose of PBS. Dosing was performed once weekly for a total of one dose. Tumor volume was measured twice weekly using a vernier caliper after dosing and calculated using the following formula: TV = (length * width²) / 2.

[0523] FIG30 shows the in vivo tumor volume-time change curve of the lung squamous cell carcinoma mouse model. The results show that compared with the negative control, Hu517-T1000-exatecan can significantly inhibit tumor growth, while UC-961-vc-MMAE does not produce any tumor inhibitory effect.

[0524] Example 13 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human non-small cell lung cancer PDX LD1-0025-200662

[0525] 6-week-old female NU / NU mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Mice were subcutaneously inoculated with non-small cell lung cancer tumors (specimen number 200662, from Xi'an Lidi Biotechnology Co., Ltd.). When the average tumor volume reached 150 mm 3 The antibody-drug conjugate ch517-T1000-exatecan prepared in Example 5 was administered intravenously at a dose of 10 mg / kg. A negative control group, Vehicle, received an equal dose of PBS intravenously. Dosing was performed once weekly (QW) for a total of two doses. Tumor volume was measured twice weekly using a vernier caliper after dosing and calculated using the following formula: TV = (length * width²) / 2.

[0526] FIG31 shows a graph of the in vivo tumor volume-time change in a non-small cell lung cancer mouse model. The results show that ch517-T1000-exatecan can significantly inhibit tumor growth compared with the negative control.

[0527] Example 14 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human non-small cell lung cancer PDX LD1-0025-200730

[0528] Six-week-old female NU / NU mice weighing 18-21 g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Mice were subcutaneously inoculated with colorectal cancer tumors (specimen number 200730, from Xi'an Lidi Biotechnology Co., Ltd.). When the average tumor volume reached 150 mm 3 The antibody-drug conjugates Hu517-T1000-exatecan and UC-961-vc-MMAE prepared in Example 5 were administered intravenously at doses of 2.5 mg / kg and 5 mg / kg, respectively. A negative control group, Vehicle, received an equal dose of PBS intravenously. Dosing was performed once weekly (QW) for a total of two doses. Tumor volume was measured twice weekly after dosing using a vernier caliper and calculated using the following formula: TV = (length * width²) / 2.

[0529] Figure 32 shows the in vivo tumor volume-time change curve of the non-small cell lung cancer mouse model. The results show that compared with the negative control, Hu517-T1000-exatecan can significantly inhibit tumor growth, while UC-961-vc-MMAE does not produce any tumor growth inhibitory effect.

[0530] Example 15 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human triple-negative breast cancer PDX LD1-2009-362263

[0531] Six-week-old female NU / NU mice weighing 18-21 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were subcutaneously inoculated with triple-negative breast cancer tumors (specimen number 362263, from Xi'an Lidi Biotechnology Co., Ltd.). When the average tumor volume reached 150 mm 3 The antibody-drug conjugates Hu517-T1000-exatecan and UC-961-vc-MMAE prepared in Example 5 were administered intravenously at doses of 10 mg / kg and 5 mg / kg, respectively. A negative control group, Vehicle, received an equal dose of PBS intravenously. Dosing was performed once weekly (QW) for a total of two doses. Tumor volume was measured twice weekly after dosing using a vernier caliper and calculated using the following formula: TV = (length * width²) / 2.

[0532] Figure 33 shows a graph of tumor volume over time in a triple-negative breast cancer mouse model. The results demonstrate that Hu517-T1000-exatecan significantly inhibited tumor growth compared to the negative control, while UC-961-vc-MMAE only partially inhibited tumor growth. Given the different tolerance doses of the two agents, T1000-exatecan has a higher tolerance dose, typically more than five times that of vc-MMAE. Therefore, Hu517-T1000-exatecan of the present invention has a larger therapeutic window.

[0533] The present disclosure describes a plurality of embodiments, but this description is exemplary rather than restrictive, and it will be apparent to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present disclosure. Although many possible feature combinations are shown herein and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with any other feature or element in any other embodiment, or may replace any other feature or element in any other embodiment.

Claims

1. An antibody or an antigen-binding fragment thereof capable of specifically binding to ROR1, comprising: (1)(a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL; or (2)(a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) the sequences of CDR1, CDR2 and CDR3 contained in VH as shown in SEQ ID NO: 37 or 41; and / or The CDR1, CDR2 and CDR3 sequences contained in the VL as shown in SEQ ID NO: 38 or 42; or (2) the sequences of CDR1, CDR2 and CDR3 contained in VH as shown in SEQ ID NO: 39; and / or The CDR1, CDR2 and CDR3 sequences contained in VL as shown in SEQ ID NO:40; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2, or a sequence consisting of the following sequence: SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions compared to SEQ ID NO:

45. A sequence containing a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of one or more amino acids), and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 4, or consisting of the following sequence: SEQ ID NO:46, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO:46, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO: 2; and VH CDR3 as shown in SEQ ID NO: 3; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 4; VL CDR2 as shown in SEQ ID NO: 5; and VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 1; a VH CDR2 as shown in SEQ ID NO: 45; and a VH CDR3 as shown in SEQ ID NO: 3; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 4; a VL CDR2 as shown in SEQ ID NO: 5; and a VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO: 2; and VH CDR3 as shown in SEQ ID NO: 3; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 46; VL CDR2 as shown in SEQ ID NO: 5; and VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 1; a VH CDR2 as shown in SEQ ID NO: 45; and a VH CDR3 as shown in SEQ ID NO: 3; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 46; a VL CDR2 as shown in SEQ ID NO: 5; and a VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO:2; and, VH CDR3 as shown in SEQ ID NO:3; and, VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO: 45; and VH CDR3 as shown in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 4; VL CDR2 as shown in SEQ ID NO: 5; and VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO: 2; and VH CDR3 as shown in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 46; VL CDR2 as shown in SEQ ID NO: 5; and VL CDR3 as shown in SEQ ID NO: 6; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1; VH CDR2 as shown in SEQ ID NO: 45; and VH CDR3 as shown in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 46; VL CDR2 as shown in SEQ ID NO: 5; and VL CDR3 as shown in SEQ ID NO: 6; or (2)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 10, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10, or a sequence consisting of the following sequence: SEQ ID NO: 47, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 47, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 11, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 7; a VH CDR2 as shown in SEQ ID NO: 8; and a VH CDR3 as shown in SEQ ID NO: 9; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 10; a VL CDR2 as shown in SEQ ID NO: 11; and a VL CDR3 as shown in SEQ ID NO: 12; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 7; a VH CDR2 as shown in SEQ ID NO: 8; and a VH CDR3 as shown in SEQ ID NO: 9; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 47; a VL CDR2 as shown in SEQ ID NO: 11; and a VL CDR3 as shown in SEQ ID NO: 12; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 7; VH CDR2 as shown in SEQ ID NO: 8; and VH CDR3 as shown in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 10; VL CDR2 as shown in SEQ ID NO: 11; and VL CDR3 as shown in SEQ ID NO: 12; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 7; VH CDR2 as shown in SEQ ID NO: 8; and VH CDR3 as shown in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 47; VL CDR2 as shown in SEQ ID NO: 11; and VL CDR3 as shown in SEQ ID NO: 12; or (3)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 13, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 14, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 14, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 15; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 16, (v) a VL CDR2 consisting of SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 17, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 18; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 13; a VH CDR2 as shown in SEQ ID NO: 14; and a VH CDR3 as shown in SEQ ID NO: 15; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 16; a VL CDR2 as shown in SEQ ID NO: 17; and a VL CDR3 as shown in SEQ ID NO: 18; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 13; VH CDR2 as shown in SEQ ID NO: 14; and VH CDR3 as shown in SEQ ID NO: 15; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 16; VL CDR2 as shown in SEQ ID NO: 17; and VL CDR3 as shown in SEQ ID NO: 18; or (4)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 19, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 20, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 21; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 22, or having a a sequence in which one or more amino acids are substituted, deleted or added (e.g., 1 or 2 amino acids are substituted, deleted or added), (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 23, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 19; a VH CDR2 as shown in SEQ ID NO: 20; and a VH CDR3 as shown in SEQ ID NO: 21; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 22; a VL CDR2 as shown in SEQ ID NO: 23; and a VL CDR3 as shown in SEQ ID NO: 24; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 19; VH CDR2 as shown in SEQ ID NO: 20; and VH CDR3 as shown in SEQ ID NO: 21; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 22; VL CDR2 as shown in SEQ ID NO: 23; and VL CDR3 as shown in SEQ ID NO: 24; or (5)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 25, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 26, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 27; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 28, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 29, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 30; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 25; VH CDR2 as shown in SEQ ID NO: 26; and VH CDR3 as shown in SEQ ID NO: 27; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 28; VL CDR2 as shown in SEQ ID NO: 29; and VL CDR3 as shown in SEQ ID NO: 30; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 25; VH CDR2 as shown in SEQ ID NO: 26; and VH CDR3 as shown in SEQ ID NO: 27; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 28; VL CDR2 as shown in SEQ ID NO: 29; and VL CDR3 as shown in SEQ ID NO: 30; or (6)(a) the following three heavy chain variable region (VH) CDRs: (i) a VH CDR1 consisting of the following sequence: SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 31, (ii) a VH CDR2 consisting of the following sequence: SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 32, and (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 33, or a sequence having one or more amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 33; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the following sequence: SEQ ID NO: 34, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 34, (v) a VL CDR2 consisting of the following sequence: SEQ ID NO: 35, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 35, and (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 36; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:31; VH CDR2 as shown in SEQ ID NO:32; and VH CDR3 as shown in SEQ ID NO:33; and / or, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:34; VL CDR2 as shown in SEQ ID NO:35; and VL CDR3 as shown in SEQ ID NO:36; Preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:31; VH CDR2 as shown in SEQ ID NO:32; and VH CDR3 as shown in SEQ ID NO:33; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:34; VL CDR2 as shown in SEQ ID NO:35; and VL CDR3 as shown in SEQ ID NO:

36.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: (1)(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 37; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 37; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:37; and (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 38; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 38; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:38; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 37 or a sequence similar to SEQ ID NO: 37 : a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:38, and a light chain variable region (VL) comprising a sequence having a sequence as shown in SEQ ID NO:38 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:38; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO:37 and a VL having the sequence shown in SEQ ID NO:38; or (2)(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 39; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 39; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:39; and (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 40; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 40; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:40; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO:39, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:39, and a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO:40, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

40. Sequence of sequence identity; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO:39 and a VL having the sequence shown in SEQ ID NO:40; or (3)(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 41; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 41; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:41; and (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 42; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 42; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:42; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising a sequence as set forth in SEQ ID NO:41, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO:41, and a light chain variable region (VL) comprising a sequence as set forth in SEQ ID NO:42, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO:X; Preferably, the antibody or antigen-binding fragment thereof comprises: A VH having the sequence shown in SEQ ID NO:41 and a VL having the sequence shown in SEQ ID NO:

42.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is humanized; Preferably, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin; Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and / or a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene); Preferably, the heavy chain framework region and / or the light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to mouse residues.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a constant region derived from human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3 or IgG4); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, κ or λ).

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a mouse antibody, a rabbit antibody, a humanized antibody, a fully human antibody, a chimeric antibody (e.g., a human-mouse chimeric antibody), a bispecific antibody, a multispecific antibody, a single-chain antibody, a dAb, a complementarity determining region fragment, Fv, a single-chain Fv (scFv), Fd, Fab, Fab' and F(ab')2, Preferably, the monoclonal antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which has a concentration of less than or equal to 1.0×10 -8 M of K D Binds human or monkey ROR1.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region as described in any one of claims 1 to 8, or encoding the antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region as described in any one of claims 1 to 8.

10. A vector comprising the isolated nucleic acid molecule of claim 9.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of an antibody-drug conjugate, or in the preparation of a kit for diagnosing a disease, especially a ROR1-mediated disease or condition, or in the preparation of a medicament for preventing and / or treating a disease, especially a ROR1-mediated disease or condition.

13. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in formula (I), in: Q1-L1-L2-L3 is a linker, Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is coupled to Q1 in the linker via the S atom in the antibody or antigen-binding fragment thereof, T is a drug unit or a diagnostic agent unit, which is coupled to L3 in the linker through an N atom or an O atom in the drug unit or the diagnostic agent unit, m is a number between 1 and 9, for example, m is a number between 2 and 9, for example, about 4, about 5, about 6, about 7, about 8, or about 9, Q1 is selected from: Replaced Replaced Replaced and replaced by L1 is -L 1a -C(=O)-, where L 1a is selected from the group consisting of an alkylene group, a polyethylene glycol group, an alkenylene group, an alkynylene group, an alicyclic group, an alicyclic heterocyclic group, an arylene group, a heteroarylene group, a substituted alkylene group, a substituted polyethylene glycol group, a substituted alkenylene group, a substituted alkynylene group, a substituted alicyclic group, a substituted alicyclic heterocyclic group, a substituted arylene group, and a substituted heteroarylene group, L2 is a polypeptide residue or a substituted polypeptide residue, L3 or replaced 14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein L2 and / or L3 may be substituted with CH2NHCH3, a polysarcosine residue, a glycosylated polyethylene glycol fragment, a substituted polysarcosine residue or a substituted glycosylated polyethylene glycol fragment.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 13, wherein the polypeptide residue comprises an amino acid selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of claim 13, wherein the polypeptide residue is selected from the group consisting of phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein L 1a -(CH2) n1 -, n1 is a number between 1 and 10, for example, a number between 1 and 8, a number between 1 and 6, or a number between 1 and 4.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13 or 14, wherein L3 is substituted with a polysarcosine residue, a glycosylated polyethylene glycol fragment, a substituted polysarcosine residue or a substituted glycosylated polyethylene glycol fragment. The polysarcosine residue is connected to the benzene ring through the structural unit -X-. The structural unit -X- is or replaced The substituted polysarcosine residue is wherein n2 is a number between 4 and 18, for example, a number between 4 and 12, and R is selected from: C1-C6 alkyl, C1-C6 cycloalkyl and C1-C6 alkoxy, The substituted glycosylated polyethylene glycol fragment is Here, n3 is a number between 4 and 18, for example, a number between 4 and 12.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein the drug unit is selected from: (i) Tubulin inhibitors, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin or its analogs (e.g., tubulysin A, tubulysin B, tubulysin D, tubulysin H, tubulysin U, tubulysin V), eribulin or its analogs, vinblastine or its analogs (e.g., vincristine, vindesine, vinblastine, DAVLBH), Aur0101; (ii) Camptothecin topoisomerase inhibitors, such as Exatecan (CAS 171335-80-1), DXd (CAS 1599440-33-1), 7-ethyl-10-hydroxycamptothecin (SN38, CAS 86639-52-3), Belotecan (CAS 213819-48-8), (4-NH2)-Exatecan (AZD'0132, CAS 2495742-21-5), 7-MAD-MDCPT (CAS 765871-81-6), 7-Aminomethyl-10-methyl-11-fluoro camptothecin (CAS 2378616-23-8); (iii) Immune activators, such as TLR7 / 8 agonists and STING agonists.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein Q1-L1-L2-L3-T is selected from:

21. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein the antibody-drug conjugate is selected from: wherein m and Ab are as defined in claim 13.

22. A method for preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 13 to 21, comprising: Reduction of Ab to obtain Ab'; Ab' is coupled with the compound represented by formula LD to obtain the antibody-drug conjugate represented by formula (I), wherein: Ab, Q1, L1, L2, L3, T and m are as defined in any one of claims 13 to 21.

23. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 13 to 21, and one or more pharmaceutically acceptable carriers and / or excipients.

24. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 13 to 21, or the pharmaceutical composition according to claim 23, in the preparation of a medicament for preventing and / or treating a disease, especially a ROR1-mediated disease or condition, or in the preparation of a medicament for treating and / or preventing a tumor, or in the preparation of a kit for diagnosing a disease, especially a ROR1-mediated disease or condition.

25. The use of claim 24, wherein the ROR1-mediated disease or condition is a cancer in which ROR1 is highly expressed, Preferably, the cancer with high expression of ROR1 is selected from head and neck squamous cell carcinoma, lung cancer (such as non-small cell lung cancer, including lung adenocarcinoma and lung squamous cell carcinoma), urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer (such as triple-negative breast cancer), bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, carcinoma, thyroid cancer, penile cancer, melanoma, leukemias (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL)), malignant lymphomas (e.g., mantle cell lymphoma (MCL)), plasmacytomas, myelomas, and sarcomas.

26. The use of claim 24, wherein the tumor is selected from the group consisting of head and neck squamous cell carcinoma, lung cancer, urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumors, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, melanoma, leukemia, malignant lymphoma, plasmacytoma, myeloma and sarcoma; Preferably, the tumor is selected from the group consisting of head and neck squamous cell carcinoma, pancreatic cancer, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma and colorectal cancer; Preferably, the tumor is selected from head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung cancer and triple-negative breast cancer; Preferably, the non-small cell lung cancer is selected from lung adenocarcinoma and lung squamous cell carcinoma.

27. A kit or composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the isolated nucleic acid molecule according to claim 9, the vector according to claim 10, the host cell according to claim 11, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 13 to 21, or the pharmaceutical composition according to claim 23.

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