WO2024193605A1 - Ror1-targeting antibody, antibody-drug conjugate comprising same, preparation method therefor, and use thereof - Google Patents

Abstract

Provided are a ROR1-targeting antibody, an antibody-drug conjugate comprising same, a preparation method therefor, and a use thereof. The ROR1-targeting antibody or an antigen-binding fragment thereof has a heavy chain variable region, a light chain variable region, and a complementarity determining region (CDR). Additionally provided are an antibody-drug conjugate comprising the ROR1-targeting antibody or an antigen-binding fragment thereof, a preparation method therefor, and a use thereof.

Description

Antibodies targeting ROR1, antibody-drug conjugates containing the same, preparation methods and uses

This application claims the priority of the Chinese patent application filed with the China Patent Office on March 20, 2023, with application number 202310271589.8 and invention name “Antibodies targeting ROR1, antibody-drug conjugates containing the same, preparation methods and uses”, the contents of which should be understood to be incorporated into this application by reference.

Technical Field

The present invention generally relates to the field of medicine, and in particular to an antibody targeting ROR1, an antibody-drug conjugate containing the same, a preparation method and uses thereof.

Background Art

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type I transmembrane receptor tyrosine kinase protein that is highly expressed during early embryonic development and plays an important role in a variety of physiological processes, including regulating cell division, proliferation, migration, etc., and participating in the formation of organs such as nerves, bones and blood vessels. In the subsequent fetal development, the expression of ROR1 gradually decreases, and there is basically no expression of ROR1 on the surface of normal children and adult tissue cells (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 hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL), and the expression of ROR1 is closely related to the progression of the disease and the therapeutic effect (Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071; Front Oncol. 2021 May 28; 11: 680834). For example, the proportion of ROR ⁺ in CLL patients exceeds 90% (Blood. 2021 Jun 17; 137(24): 3365-3377.). The expression of ROR1 in invasive malignant tumors initiates a transcriptional process similar to embryonic development. ROR1 can bind to wnt5a, participate in the signal transduction of the wnt pathway, and interact with signal pathways such as EGFR and Met to promote the growth, proliferation and metastasis of tumor cells. ROR1 plays an important role in the development of cancer stem cells and the epithelial-mesenchymal transition (EMT) process. Cancer stem cells are cancer cells with more stem cell characteristics in tumors, and they are usually more resistant to chemotherapy drugs. The EMT process transforms the cell morphology from epithelial cells to mesenchymal cells, making them more invasive and promoting cancer metastasis.

The role of ROR1 in lung cancer includes inhibiting apoptosis, enhancing EGFR signal transduction and inducing proliferation. At the same time, there are many papers that explain that ROR1 plays an important role in the metastasis of lung adenocarcinoma. Targeted therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) plays a very important role in the comprehensive treatment of non-small cell lung cancer (NSCLC). However, almost all patients inevitably develop drug resistance after a period of use. EGFR T790M mutation and elevated ROR1 expression were found in resistant patients, suggesting that ROR1-targeted therapy may be applicable to tumor patients with EGFR mutation and EGFR TKI resistance (Transl Lung Cancer Res. 2014 Jun; 3(3):122-30.).

In summary, ROR1 can be used as a specific tumor marker and tumor therapeutic drug target for drug development.

Summary of the invention

Antibody-drug conjugates (ADC) can be composed of antibodies (Antibody) targeting tumor-specific antigens or tumor-associated antigens and different numbers of cytotoxic drugs (Payload) coupled through linkers. It is one of the fastest-growing drug categories in oncology in recent years. Due to the dual advantages of high targeting of monoclonal antibody drugs and high activity of cytotoxic drugs in tumor tissues, ADC drugs can efficiently kill tumor cells, have fewer side effects than chemotherapy drugs, and have better efficacy than traditional antibody tumor drugs. They are called "biological missiles" in the field of tumor treatment. So far, it is reported that 14 ADC drugs have been approved for marketing worldwide, 7 for hematological tumors and 7 for the treatment of solid tumors. The targets involve CD33, CD30, CD22, CD79b, HER2, Nectin-4, Trop-2, BCMA, CD19 and TF. Among them, 4 ADC drugs are listed in China.

Currently, there is no ROR1-targeting ADC on the market. Clinically available ROR1 ADCs include: VLS-101 (VLS101), co-developed by Merck & Co. (MSD) and VelosBio, which is currently being used to treat blood cancers and solid tumors in a Phase 1 clinical trial and a Phase 2 clinical trial, respectively; LCB71, a ROR1 ADC co-developed by CStone Pharmaceuticals and LegoChem Biosciences, which carries a tumor-activated pyrrolobenzodiazepine (PBD) protoxin, and PBD, due to its own toxicity, has a narrow safety window in the treatment of solid tumors (J Thorac Oncol. 2021 Sep; 16 (9): 1429-1433.); In addition, the ROR1 iADC NBE-002 developed by Boehringer Ingelheim/NBE Therapeutics uses a specific reaction mediated by Sortase A to couple PNU-159682 to the C-termini of the heavy and light chains of the antibody in a fixed and quantitative manner. PNU-159682 has the same safety risks as PBD toxins. ROR1 is different from other ADC antigens, such as HER2, which has hundreds of thousands of copies on tumor cells. The expression level of ROR1 in most tumor cells is not very high, with no more than 5,000 copies per cell. Therefore, there is an urgent need in this field to develop a new type of antibody-drug conjugate targeting ROR1, which can increase the drug concentration of ADC drugs in tumor cells and reduce the overall drug concentration in plasma, thereby improving the therapeutic effect of ADC drugs on tumors and reducing the side effects of toxin molecules.

In order to solve the problems existing in the prior art, the first aspect of the present application provides an anti-ROR1 antibody or an antigen-binding fragment thereof, comprising:

A heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 17 and 33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 19 and 35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 5, 21 and 37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 7, 23 and 39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 9, 25 and 41, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 11, 27 and 43, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) comprising the amino acid sequence of SEQ ID NO: 7 or having at least 80%, 85%, 90%, 95%, a complementarity determining region VL CDR1 having an amino acid sequence with 98%, 99% or 100% identity;

e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(2) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(3) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) comprising the amino acid sequence of SEQ ID NO: 37 or having at least 80%, 85%, 90%, 95%, a complementarity determining region VH CDR3 having an amino acid sequence that is 98%, 99% or 100% identical to the amino acid sequence of the polypeptide; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:14, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

(2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:46 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14;

(2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:30; or

(3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:15, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and the amino acid sequence set forth in SEQ ID NO:16, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence set forth in SEQ ID NO:15 and the light chain amino acid sequence set forth in SEQ ID NO:16.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence set forth in SEQ ID NO:31 and the light chain amino acid sequence set forth in SEQ ID NO:32.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence set forth in SEQ ID NO:47 and the light chain amino acid sequence set forth in SEQ ID NO:48.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, Fv, a single chain Fv (scFv), Fd, Fab, Fab', and F(ab') ₂ .

In some embodiments, the antibody or antigen-binding fragment thereof binds human ROR1 with a KD of 2.0×10 ⁻⁹ M or less.

In some embodiments, the antibody or antigen-binding fragment thereof does not cross-react with mouse ROR1.

The second aspect of the present application provides a nucleic acid sequence encoding the above-mentioned antibody or antigen-binding fragment thereof.

In some embodiments, the nucleic acid sequence encoding the above-mentioned antibodies or their antigen-binding fragments comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 18, 20, 22, 24, 26, 28, 34, 36, 38, 40, 42 or 44.

The third aspect of the present application provides a vector or plasmid comprising a nucleic acid sequence encoding the above-mentioned antibody or antigen-binding fragment thereof.

The fourth aspect of the present application provides a host cell comprising and expressing the above-mentioned nucleic acid sequence, plasmid or vector.

The fifth aspect of the present application provides an antibody-drug conjugate having a structure shown in Formula I

Ab-(LD) _n (Formula I),

wherein Ab is any of the above antibodies or antigen-binding fragments thereof;

L is a linker;

D is a cytotoxic drug; and

n is a number between 1-10, preferably a value between 1-8; preferably n is 1, 2, 3, 4, 5, 6, 7, 8, and any value between any two values.

Further, L is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linker, disubstituted maleimide, and includes one or more linkers selected from valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

Wherein D is selected from the following group:

(i) Tubulin inhibitors, such as maytansine derivative DM1, maytansine derivative DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

(ii) Toxins targeting DNA, such as duocarmycin and pyrrolobenzodiazepine (PBD);

(iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

The present application also provides a kit for diagnosing a disease, wherein the kit comprises the anti-ROR1 antibody or antigen-binding fragment thereof of the present application, and instructions for use.

The present application also provides a composition comprising the above-mentioned antibody-drug conjugate.

In another aspect, the present application provides use of the anti-ROR1 antibody or antigen-binding fragment thereof of the present application in preparing a kit for diagnosing a disease.

The present application also provides use of the antibody-drug conjugate described herein or a composition comprising the same in the preparation of a drug for treating cancer.

In another aspect, the present application provides a method for treating a disease, comprising administering the antibody-drug conjugate or composition described herein to a subject in need thereof.

The present application also provides a method for diagnosing a disease, comprising using the anti-ROR1 antibody or antigen-binding fragment thereof of the present application or the kit described above.

Other features and advantages of the present application will be described in the following description and will become apparent in part from the description. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein. The present invention is also provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows the EC ₅₀ and representative binding curves of anti-ROR1 antibodies binding to human ROR1, indicating that these clones specifically bind to human ROR1;

Figure 2 shows representative binding curves of anti-ROR1 antibodies to murine ROR1, indicating that these clones did not bind to mouse ROR1.

Figure 3 shows representative binding curves of anti-ROR1 antibodies to human ROR2, indicating that ROR1-14 and ROR1-12 bind to human ROR2.

Figures 4 to 6 show the mass spectrometry deglycosylated spectrum of the humanized antibody ROR1-4 (Figure 4) and the HIC (Figure 5) and mass spectrometry deglycosylated spectrum (Figure 6) of its antibody conjugate ROR1-4-BL20MMAE (abbreviated as ROR1-4-BL20E).

Figures 7 to 9 show the mass spectrometry after deglycosylation of the humanized antibody ROR1-12 (Figure 7) and the HIC (Figure 8) and mass spectrometry after deglycosylation (Figure 9) of its antibody conjugate ROR1-12-BL20MMAE.

Figures 10-12 show the mass spectrometry deglycosylated spectrum of the humanized antibody ROR1-12 (Figure 10) and the HIC (Figure 11) and mass spectrometry deglycosylated spectrum (Figure 12) of its antibody conjugate ROR1-12-GGFG-Dxd (abbreviated as ROR1-12-DX).

FIG. 13 shows the effects of ROR1-12-BL20E and ROR1-12-DX on the growth of subcutaneous transplanted tumors of human mantle cell lymphoma JeKo-1.

FIG. 14 shows the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the growth of subcutaneous xenografts of human breast cancer MDA-MB-231.

FIG. 15 shows the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the body weight of mice subcutaneously transplanted with human breast cancer MDA-MB-231.

FIG. 16 shows the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the growth of subcutaneous transplanted tumors of human lung cancer H1975.

Figure 17 shows the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on human lung cancer H1975 Effects of transplantation on the body weight of mice.

Details

In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "and/or" as used herein should be considered as a specific disclosure of each of two or more specified features or components, with or without the other features or components. 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).

It is to be understood that wherever aspects are described with the language "comprising" or "including," similar aspects described with the terms "consisting of" and/or "consisting essentially of are also provided.

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 a variation may be as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of a 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.

The term "ROR1" refers to receptor tyrosine kinase-like orphan receptor 1, also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR1). The term "ROR1" includes variants, isoforms, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific for human ROR1 protein may cross-react with ROR1 proteins from non-human species (e.g., cynomolgus monkeys). In other embodiments, antibodies specific for human ROR1 protein may be completely specific for human ROR1 protein and show no cross-reactivity to other species or other types, or may cross-react with ROR1 from certain other species but not all other species.

The term "human ROR1" refers to a human ROR1 sequence, such as the amino acid sequence of human ROR1 having NCBI reference sequence number NP_005003.2.

The term "antibody" is used herein in a broad sense as understood by those of ordinary skill in the art, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as long as they show the desired antigen-binding activity. The antibody can be a complete antibody (e.g., having two full-length light chains and two full-length heavy chains) of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2). "Antibody" can also refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the entire antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') ₂ fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) an isolated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using recombinant methods through a synthetic linker that enables them to form a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv)).

The term "monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may exist in a small amount, the individual antibodies that constitute the population are identical. Monoclonal antibodies are highly specific for a single antigenic epitope. In contrast, polyclonal antibodies generally include many antibodies for different epitopes (or to their specificity). "Monoclonal" refers to the antibody characteristics from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any specific method. For example, monoclonal antibodies can be prepared by a hybridoma method, or can be prepared by a recombinant DNA method. "Monoclonal antibody" can also be separated from a phage antibody library.

The term "multispecific antibody" refers to an antibody that has binding specificities for at least two different epitopes on the same antigen or different antigens. The term "bispecific antibody" means an antibody that has binding specificities for two different epitopes.

The term "humanized antibody" as used herein refers to an antibody form comprising sequences from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies comprise minimal sequences derived from non-human immunoglobulins. In general, humanized antibodies substantially comprise at least one and usually two variable domains of all variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins.

The terms "human antibody" or "fully human antibody" include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include antibodies that contain CDR sequences that are not encoded by human germline immunoglobulin sequences (e.g., 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 in which CDR sequences derived from another mammalian species germline (e.g., mouse) are grafted onto human framework sequences (i.e., humanized antibodies). Fully human antibodies or human antibodies may be derived from transgenic mice carrying human antibody genes or from human cells.

The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of the antibody and other agents or other antibodies.

As used herein, the terms "anti-ROR1 antibody," "anti-ROR1," "ROR1 antibody," or "antibody that binds to ROR1" refer to an antibody that is capable of binding to a ROR1 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting ROR1.

As used herein, the term "epitope" refers to a portion of an antigen (e.g., ROR1) that specifically interacts with an antibody molecule. This portion (referred to herein as an epitope determinant) typically comprises elements such as amino acid side chains or sugar side chains or components thereof. Epitope determinants can be defined using methods known in the art. Some epitopes are linear epitopes, while others are conformational epitopes.

As used herein, an "isolated antibody" refers to an antibody molecule that has been separated from a component of its natural environment.

The terms "specificity", "specific binding" or "specific for" refer to the number of different types of antigens or antigenic determinants to which a particular antibody or antigen-binding fragment thereof can bind. Thus, an antibody or antigen-binding fragment thereof as defined herein is said to be "specific" for a first target or antigen when it binds to a first antigen with an affinity (as described herein and suitably expressed, for example, as a KD value) that is at least 50 times, such as at least 100 times, preferably at least 1000 times, and up to 10,000 times or more, higher than the affinity with which the amino acid sequence or polypeptide binds to another target or polypeptide. Preferably, when an antibody or antigen-binding fragment thereof is "specific" for a target or antigen, it is able to bind to the target or antigen, but not to other targets or antigens, compared to another target or antigen.

As used herein, the term " _Kassoc " or " _Ka " refers to the association rate of a particular antibody-antigen interaction, while the term " _Kdis " or " _Kd " as used herein refers to the dissociation rate of an antibody-antigen interaction.

As used herein, the term " _KD " refers to the dissociation constant, which is obtained from the ratio of _Kd to _Ka (i.e., _Kd / _Ka ) and is expressed as a molar concentration (M). The _KD value of an antibody can be determined using methods established in the art. A preferred method for determining the _KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore system. _KD is a measure of the affinity of an antibody for an antigen.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide sequences including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom, without regard to the number of generations. Progeny may not be identical to the parent cell in nucleic acid content, but may include mutations. Mutant progeny with the same function or biological activity as screened or selected in the original transformed cell are included herein.

IC ₅₀ (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It indicates the half amount of a drug or substance (inhibitor) in inhibiting a certain biological process (or certain substances involved in this process, such as enzymes, cell receptors or microorganisms). IC ₅₀ can measure the sensitivity of the antibody. The lower the IC ₅₀ , the higher the sensitivity of the antibody.

_EC50 (concentration for 50% of maximal effect) refers to the concentration that can cause 50% of the maximum effect.

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the required dosage and for the required period of time. A therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 1:1 relative to an untreated subject. 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70% and still more preferably at least about 80% or 90%.

"Percent (%) sequence identity" relative to a reference sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the residues in the reference sequence after the candidate sequence is compared with the reference sequence and, if necessary, a gap is introduced to obtain the maximum percentage sequence identity, and any conservative substitution is not considered as part of the sequence identity. Comparisons for determining percentage sequence identity can be achieved in a variety of ways in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. When referring to percentages of sequence identity in this application, unless otherwise expressly stated, these percentages are calculated relative to the full length of the longer sequence. Calculations relative to the full length of the longer sequence are applicable to both nucleic acid sequences and polypeptide sequences.

The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional components which are unacceptably toxic to a subject to which the preparation would be administered.

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, etc., as known in the art. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Each carrier should be pharmaceutically and physiologically acceptable in terms of compatibility with the other ingredients and harmless to the subject. Unless any conventional medium or agent is incompatible with the active ingredient, its administration in the therapeutic composition is envisioned.

It should be understood that the term "treat," ...

The term "immunoconjugate" as defined herein refers to an antibody according to the present disclosure or any antigen-binding fragment thereof that is conjugated or linked to an additional agent. Immunoconjugates can be prepared by any method known to those skilled in the art, for example, by cross-linking an additional agent to an antibody according to the present disclosure or by recombinant DNA methods.

Anti-ROR1 antibody or antigen-binding fragment thereof

The first aspect of the present application provides an anti-ROR1 antibody or an antigen-binding fragment thereof,

A heavy chain variable region, the heavy chain variable region comprising:

a) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 17 and 33 or having at least 80%, a complementarity determining region VH CDR1 having an amino acid sequence that is 85%, 90%, 95%, 98%, 99% or 100% identical;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 19 and 35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 5, 21 and 37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 7, 23 and 39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 9, 25 and 41, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 11, 27 and 43, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) comprising the amino acid sequence of SEQ ID NO: 9 or having at least 80%, 85%, 90%, 95%, a complementarity determining region VL CDR2 having an amino acid sequence that is 98%, 99% or 100% identical to the amino acid sequence of the polypeptide; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(2) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(3) a heavy chain variable region, the heavy chain variable region comprising:

a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

and

c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

A light chain variable region, the light chain variable region comprising:

d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and

f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region comprising a heavy chain amino acid sequence as set forth in SEQ ID NO:13, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising a light chain amino acid sequence as set forth in SEQ ID NO:14, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto;

(2) a heavy chain variable region comprising a heavy chain amino acid sequence as set forth in SEQ ID NO:29 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising a light chain amino acid sequence as set forth in SEQ ID NO:30 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or

(3) a heavy chain variable region comprising a heavy chain amino acid sequence as shown in SEQ ID NO:45 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising a light chain amino acid sequence as shown in SEQ ID NO:46 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14;

(2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:30; or

(3) a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and The amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence set forth in SEQ ID NO:31 and the light chain amino acid sequence set forth in SEQ ID NO:32.

In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence set forth in SEQ ID NO:47 and the light chain amino acid sequence set forth in SEQ ID NO:48.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, Fv, a single chain Fv (scFv), Fd, Fab, Fab', and F(ab')2.

In some embodiments, the antibody or antigen-binding fragment thereof binds human ROR1 with a KD of 2.0×10 ⁻⁹ M or less.

In some embodiments, the antibody or antigen-binding fragment thereof does not cross-react with mouse ROR1.

Table 1 Sequence composition of antibodies

Protein expression

Molecular biological techniques suitable for producing the polypeptides or proteins (eg, antibodies or antigen-binding fragments thereof) of the present application in cells are well known to those of ordinary skill in the art.

A polypeptide or protein can be expressed from a polynucleotide sequence. The polynucleotide sequence can be contained in a vector present in the cell, or can be incorporated into the genome of the cell.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide sequences including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

The term "vector" as used herein refers to an oligonucleotide molecule (DNA or RNA) used as a carrier to transfer exogenous genetic material into a cell. A vector may be an expression vector for expressing genetic material in a cell. Such vectors may include a promoter sequence operably connected to a polynucleotide sequence encoding a gene sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer and stop codon known in the art may be used to express polypeptides from the vector of the present application. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g., yeast artificial chromosomes).

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the present application. The cell can be a prokaryotic or eukaryotic cell. Suitable prokaryotic cells include Escherichia coli cells. Examples of suitable eukaryotic cells include yeast cells, plant cells, insect cells, or mammalian cells (e.g., Chinese hamster ovary (CHO) cells). In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow the same post-translational modifications as eukaryotic cells. In addition, Very high expression levels are possible in eukaryotes, and the use of appropriate tags makes it easier to purify proteins from eukaryotes. Specific plasmids can also be used, which enhance the secretion of proteins into the culture medium. Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cells within cultured plant tissues or animal tissues.

The method of producing the target polypeptide can include culturing or fermenting the modified cells to express the polypeptide. The culturing or fermentation can be carried out in a bioreactor with an appropriate supply of nutrients, air/oxygen and/or growth factors. The secreted protein can be collected by separating the cells from the culture medium/fermentation broth, extracting the protein and isolating the individual proteins to separate the secreted polypeptide. Cultivation, fermentation and separation techniques are well known to those of ordinary skill in the art.

A bioreactor includes one or more containers in which cells can be cultured. Cultivation in a bioreactor can be continuous, with reactants flowing in continuously and cultured cells flowing continuously from the reactor. Alternatively, cultivation can be carried out in batches. Bioreactors monitor and control environmental conditions, such as pH, oxygen, inflow and outflow rates, and agitation within the container, to provide optimal conditions for the cultured cells.

After culturing cells expressing the target polypeptide/protein, the polypeptide/protein is preferably separated. Any suitable method for separating polypeptides/proteins from cell cultures known to those of ordinary skill in the art can be used. In order to separate the target polypeptide/protein from the culture, it may be necessary to first separate the cultured cells from the culture medium containing the target polypeptide/protein. If the target polypeptide/protein is secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide/protein by centrifugation. If the target polypeptide/protein aggregates in the cells, the cells must be destroyed before centrifugation, for example, using ultrasonic treatment, rapid freeze-thaw or osmotic lysis. Centrifugation will produce a precipitate containing cultured cells or cell fragments of cultured cells and a supernatant containing the culture medium and the target polypeptide/protein.

It may then be necessary to separate the polypeptide/protein of interest from the supernatant or culture medium, which may contain other proteins and non-protein components. A common method for separating polypeptide/protein components from supernatants and culture medium is by precipitation. Polypeptides/proteins of different solubility are precipitated in different concentrations of a precipitant such as ammonium sulfate. For example, at low concentrations of a precipitant, water-soluble polypeptides/proteins are extracted. Thus, by adding increasing concentrations of a precipitant, polypeptides/proteins of different solubility can be distinguished. Chromatography can then be used to remove the ammonium sulfate from the separated polypeptides/proteins.

Other methods for separating different polypeptides/proteins are known to those of ordinary skill in the art, such as ion exchange chromatography and size chromatography. These can be used as an alternative to precipitation, or can be performed after precipitation.

Once the polypeptide/protein of interest is isolated from the culture, it may be necessary to concentrate it. Many methods for concentrating the polypeptide/protein of interest are well known to those of ordinary skill in the art, such as ultrafiltration or freeze drying.

Immunoconjugates

An example of an immunoconjugate is an antibody-drug conjugate, also known as an antibody-drug conjugate or antibody-drug conjugate. Antibody-drug conjugates are made by connecting a monoclonal antibody targeting a specific antigen to a small molecule cytotoxic drug through a linker, combining the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. ADC consists 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 the payload.

The present application provides a novel class of antibody-drug conjugates having the formula Ab-(LD) _n , wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase-like orphan receptor 1 (ROR1); L is a linker; D is a cytotoxic drug; and n is an integer from 1 to 10.

Linker

According to the mechanism of drug release in cells, “linkers” or “linkers of antibody drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.

For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of the small molecule drug, linker, and antibody amino acid residues. The resulting change in the drug molecular structure does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect).

As the name implies, cleavable linkers can be cleaved in target cells and release active drugs (small molecule drugs themselves). Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzyme-labile linkers. Chemically unstable linkers can be selectively cleaved due to different properties of plasma and cytoplasm. Such properties include pH, glutathione concentration, etc. Linkers that are sensitive to pH are usually called acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH5.0-6.5) and lysosomes (pH4.5-5.0). The first generation of antibody-drug conjugates mostly use this type of linker, such as hydrazone, carbonate, acetal, ketal. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers in the new generation of antibody-drug conjugates to a certain extent.

Glutathione-sensitive linkers are also called disulfide linkers. Drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration in the blood (micromolar range). This is especially true for tumor cells, whose low oxygen content leads to increased reductase activity and thus higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have good stability in plasma.

Enzyme-labile linkers, such as peptide linkers, allow for better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (the levels of such enzymes are increased in some tumor tissues). This peptide linkage is considered to be very stable in plasma circulation because proteases are generally inactive due to the unfavorable extracellular pH and serum protease inhibitors. Enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates in view of their high plasma stability and good intracellular cleavage selectivity and effectiveness. Typical enzyme-labile linkers include Val-Cit (VC), Phe-Lys, etc.

The self-releasing linker is generally embedded between the cleavable linker and the active drug, or is itself a part of the cleavable linker. The mechanism of action of the self-releasing linker is that when the cleavable linker is cleaved under appropriate conditions, the self-releasing linker can spontaneously rearrange its structure and release the active drug connected to it. Common self-releasing linkers include p-aminobenzyl alcohol (PAB) and β-glucuronide (β-Glucuronide).

The present application provides a linker or coupling reagent comprising a diarylthiomaleimide unit and a coupling group. The diarylthiomaleimide unit is used to cross-link the sulfhydryl groups between antibody chains (after reduction), and the coupling group is used to couple with a small molecule drug or a drug-linker unit. Due to the bidentate binding of the diarylthiomaleimide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody, these ADCs are homogeneous and have greater stability than ADCs containing monodentate linkers. Therefore, they will have an increased half-life in vivo, reduce the amount of cytotoxins released systemically, and have safer drug properties than ADCs with monodentate linkers.

In another aspect, the produced drug-linker unit is coupled to the antibody through the linker to generate a partially cross-linked conjugate. Compared with traditional antibody-drug conjugates, the drug/antibody ratio (DAR) distribution of the antibody-drug conjugate prepared by the method of the present application is narrower, thereby greatly improving the uniformity of the product and the uniformity of the pharmacological properties. The antibody-drug conjugate can be used for targeted delivery of drugs to target cell populations, such as tumor cells. Antibody-drug conjugates can specifically bind to cell surface proteins, and the resulting conjugate is then endocytosed by the cell. In the cell, the drug is released as an active drug to produce efficacy. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that can bind to antigens; or antibody Fc fusion proteins. "Drugs" are highly active drugs, and in some cases, the linker can be polyethylene glycol.

In one embodiment, the linker L can be a cleavable linker or a non-cleavable linker. In one embodiment, the linker is a chemical linker. In one embodiment, the linker comprises a covalent bond, such as an ester bond, an ether bond, an amine bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphorus ester bond, a peptide bond, a hydrazone bond, or a combination thereof. In one embodiment, the linker comprises a hydrophobic poly (ethylene glycol) linker. In one embodiment, the linker comprises a peptide bond.

In one embodiment, the cytotoxic drug D can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

Useful cytotoxic drug D classes may include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of particularly useful cytotoxic drug D classes include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBD), indolinobenzodiazepines and oxazolidinobenzodiazepines), and vinca alkaloids.

In one embodiment, the cytotoxic drug D can be selected from a chemotherapeutic agent, a growth inhibitor, a calicheamicin drug unit, an antimitotic agent, a radioisotope, or a combination thereof. In one embodiment, the cytotoxic drug D comprises calicheamicin, ozogamicin, monomethyl auristatin E, emtansine, 7-ethyl-10-hydroxycamptothecin (SN-38), exatecan, a derivative thereof, or a combination thereof. In one embodiment, the cytotoxic drug D comprises monomethyl auristatin E, emtansine, 7-ethyl-10-hydroxycamptothecin (SN-38), exatecan, α-amanitin, duocarmycin, pyrrolobenzodiazepine (PBD), PNU-159682 and pharmaceutically acceptable salts, esters and analogs thereof.

Other common LD moieties of antibody-drug conjugates are e.g.

In some preferred embodiments, the linker L is a maleimide linker; preferably, L is a disubstituted maleimide linker. The linker can be fully/partially cross-coupled to the cysteine thiol groups of the light chain-heavy chain and heavy chain-heavy chain disulfide bonds of the antibody, and the ROR1-targeted antibody-drug conjugate obtained by this coupling method has a relatively more uniform drug/antibody ratio (DAR) distribution compared to traditional antibody-drug conjugates. The structure of the ROR1 antibody-drug conjugate with the disubstituted maleimide linker is shown in Formulas Ia and Ib:

in,

Ar' is selected from the group consisting of a substituted or unsubstituted C6-C10 arylene group, a substituted or unsubstituted 5-12 membered heteroarylene group;

L ₁ is -O(CH ₂ CH ₂ O) _n - connected to the Ar' group, wherein n is selected from any integer from 1 to 20; preferably any integer from 1 to 10;

_L2 is a chemical bond or an AA-PAB structure; wherein AA is a polypeptide fragment consisting of 2-4 amino acids, and PAB is p-aminobenzylcarbamoyl;

CTD is a cytotoxic drug and/or a drug for treating cancer bonded to _L2 via an amide bond;

m is 1.0-5.0, preferably 3.0-4.2; more preferably 3.5-4.5; still more preferably 3.8-4.2, still more preferably 3.9-4.1, Most preferably 4.0;

Ab is an antibody or an antigen-binding fragment thereof targeting ROR1 as described in the present application.

In another preferred embodiment, the formula Ib is the product of the ring-opening of N-phenylmaleimide in formula Ia.

In another preferred embodiment, the conjugate is covalently linked to one or more drug components.

In another preferred embodiment, in the conjugate, the antibody and the drug are coupled by covalent means (eg, by being covalently linked to a linker, respectively).

In another preferred embodiment, the closed or open maleimide group is connected to the reduced sulfhydryl group of the disulfide bond in the hinge region of the antibody.

In another preferred embodiment, the antibody-drug conjugate is prepared by reducing the disulfide bonds in the hinge region of the antibody or antibody fragment to generate a pair of cysteine residues, and reacting the thiol groups in the cysteine residues with the aryl thioether in the substituted maleimide linker-drug conjugate to obtain antibody-drug conjugate Ia and/or Ib.

In another preferred embodiment, the closed or open maleimide group is connected to a fully reduced antibody, ie, the four pairs of disulfide chains in the hinge region are fully opened, preferably m is 3.8-4.2, more preferably 3.9-4.1, and most preferably 4.0.

In another preferred embodiment, the antibody targeting ROR is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a single chain antibody, Fv, a single chain Fv (scFv), Fd, Fab, Fab' and F(ab')2.

In another preferred embodiment, the antibody fragment is an antibody Fab fragment.

In another preferred embodiment, the antibody is an antibody capable of binding to ROR1.

In another preferred embodiment, the antibody or antibody fragment targeting ROR1 is the antibody or antigen-binding fragment thereof described in the first aspect of the present application.

In another preferred embodiment, Ar' is selected from the following group: phenyl, halogenated benzene, C1-C4 alkylphenyl, C1-C4 alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl, wherein: C1-C4 alkylphenyl is further preferably 4-methylphenyl; C1-C4 alkoxyphenyl is further preferably 4-methoxyphenyl.

In another preferred embodiment, Ar' is selected from substituted or unsubstituted phenylene or pyridyl, and the substitution refers to that the hydrogen atoms on the group are replaced by one or more substituents selected from the following groups: halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile, amide.

In another preferred embodiment, the AA is selected from the group consisting of Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Phe-Lys (phenylalanine-lysine), Ala-Ala-Asn (alanine-alanine-asparagine), D-Ala-Phe-Lys (D-Alanine-Phenylalanine-Lysine), Gly-Gly-Phe-Gly (Glycine-Glycine-Phenylalanine-Glycine).

In some preferred embodiments, methods for coupling cytotoxic drugs to antibodies or antigen-binding fragments thereof targeting ROR1 via maleimide linkers are known to those skilled in the art, for example, see Chinese patent applications CN201611093699.6 and CN201711169847.2.

The antibody drug conjugate provided in the present application, although still a mixture, has a narrow DAR distribution range compared to the antibody drug conjugate obtained by traditional coupling. Its average DAR value is close to 4, close to the average DAR value (2-4) range of the best antibody drug conjugate. In addition, the antibody drug conjugate rarely contains naked antibodies (DAR=0), and this component does not work on cytotoxic killing. At the same time, the antibody drug conjugate does not contain heavy conjugates (DAR=8), which are cleared very quickly in the body relative to low DAR components. Therefore, the antibody drug conjugate provided in the present application is greatly improved in terms of homogeneity.

Preparation of ROR1 antibody-drug conjugates

The preparation route of the antibody drug conjugate is as follows. The interchain disulfide bonds of the antibody are reduced to generate 2n (n is a number between 1 and 4) thiol groups. The substituted maleimide linker-drug conjugate (compound of formula Ic) of the present application is cross-linked with the reduced antibody thiol groups to generate the corresponding antibody drug conjugate, wherein the antibody drug conjugate exists in one or two forms as shown below.

Wherein, the compound of formula Ic is selected from:

wait.

A typical preparation method includes: diluting the antibody stock solution to 2-10 mg/mL with a reaction buffer, adding 140-200 times excess molar ratio of dithiothreitol (DTT), or adding 6.0-20 times excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stirring the reaction solution at 10-35° C. for 2-48 hours. The above reaction buffer can be a buffer prepared in the following proportions: 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH ₂ PO ₄ -NaOH)/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM boric acid-borax/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9 and PBS/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9.

The above reaction solution is cooled to 0-10°C. If DTT reduction is used, excess DTT needs to be removed by desalting column or ultrafiltration after the reduction reaction is completed, and then the substituted maleimide compound (preliminarily dissolved in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethylacetamide (DMA) at 10 mg/ml) is added, and the volume proportion of the organic solvent in the total reaction solution is ensured not to exceed 15%, and the coupling reaction is stirred at 0-37°C for 2-4 hours. If TCEP reduction is used, it is also possible to directly add the substituted maleimide compound for coupling without removing the remaining TCEP.

The coupling reaction mixture is purified by gel filtration using a desalting column with sodium succinate/NaCl buffer or histidine-acetic acid/sucrose, and the peak sample is collected according to the UV280 ultraviolet absorption value, or ultrafiltration is performed several times. Then, the product is sterilized by filtration and stored at low temperature. The preferred temperature is -100°C to -20°C, and the pore size of the filter device is preferably 0.15-0.3 microns.

The DAR value of the obtained antibody drug conjugate is relatively uniform. When using the different substituted maleimide linkers (linker fragments) of the present application, the ADC product uniformity is very high (usually DAR-dominant products (such as DAR values of about 4) account for at least 60%, at least 70%, at least 80%, at least 90% or more of all ADCs). For ADCs with certain differences in DAR, if it is necessary to obtain samples with better uniformity, further separation and purification can be performed using but not limited to the following methods: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC).

The antibodies or antigen-binding portions thereof of the present application may also be encoded by oncolytic viruses or used in combination with oncolytic viruses.

Reagent test kit

The present application also provides a kit for diagnosing a disease, wherein the kit comprises the anti-ROR1 antibody or antigen-binding fragment thereof of the present application, and instructions for use.

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 test kit may be formulated for determination in a clinical analyzer that does not utilize a disposable test device. Preferably, the test kit is an in vitro diagnostic agent. As used herein, the term "in vitro diagnostic" refers to a medical device, which is a reagent, a reagent product, a calibrator, a control material, a kit, an instrument, a device, an apparatus or a 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 primarily used to provide information about physiological or pathological states or about congenital anomalies or to determine safety and compatibility with potential recipients, or to monitor therapeutic measures.

Composition

The present application also provides a composition comprising the antibody-drug conjugate of the present application and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, etc., as known in the art. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Each carrier should be pharmaceutically and physiologically acceptable in terms of compatibility with the other ingredients and harmless to the subject. Unless any conventional media or agents are incompatible with the active ingredient, their use in the therapeutic composition is contemplated.

In some embodiments, the antibody-drug conjugate of the present application can be administered as a single active pharmaceutical ingredient, or can be administered in combination with other anti-cancer drugs.

In some embodiments, the antibody drug conjugate of the present application or a composition comprising the same is administered in an effective amount or effective dose, for example, in terms of the amount of the antibody, in the range of 1 ng/kg to about 100 mg/kg body weight per day. The antibody-drug conjugate of the present application can be administered once, twice, three times or more per day for 1-4 weeks or longer.

In the present application, the "effective amount" or "effective dose" refers to an amount sufficient to affect the beneficial or desired symptoms of a disease, its complications, or pathological indicators in the course of disease development.

The antibody drug conjugate of the present application or the composition comprising the same can be administered by any conventional route, such as intravenous, intraarterial, intramuscular, intraperitoneal administration, and the like.

Treatment and diagnostic methods

On the other hand, the present application provides a method for treating or improving a subject's cancer, including administering to a subject a therapeutically effective amount of an antibody or its antigen-binding fragment of the present application.Cancer can be a blood cancer or a solid tumor, selected from lymphoma, CLL, small lymphocytic lymphoma, marginal cell B-cell lymphoma, Burkett's lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer and head and neck cancer.In some embodiments, at least one additional anti-cancer antibody can be administered together with the antibody or its antigen-binding fragment of the present application, such as anti-PD-1 antibody, anti-LAG-3 antibody and/or anti-CTLA-4 antibody.In another embodiment, the antibody or its antigen-binding portion of the present application is administered together with a cytokine (e.g., IL-2 and/or IL-21) or a costimulatory antibody (e.g., anti-CD137 and/or anti-GITR antibody). The antibodies of the present application can be, for example, mouse, human, chimeric or humanized antibodies.

On the other hand, the present application provides a method for diagnosing or prognosing a subject's cancer, comprising collecting a tissue sample of interest from a subject, and contacting the tissue sample with an antibody or its antigen-binding portion thereof. If a certain amount of ROR1 is detected, the subject may be diagnosed with cancer, and an increase/decrease in ROR1 expression indicates cancer development/improvement. The cancer may be a blood cancer or a solid tumor selected from lymphoma, CLL, small lymphocytic lymphoma, marginal cell B-cell lymphoma, Burkett's lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.

Example

Example 1: Phage library panning and screening of ROR1 monoclonal antibodies

Antibody single-chain phage display was generated by cloning a repertoire of light chain variable regions (VL) and heavy chain variable regions (VH) The heavy chain library and the light chain library were generated by PCR amplification from human lymphocytes collected mainly from peripheral blood and neonatal umbilical cord blood. The VL library and the VH library were mixed and PCR was performed using overlapping primers. The final form of the antibody is a single-chain Fv (scFv), in which the VH fragment and the VL fragment are connected by a flexible linker peptide (SGGSTITSYNVYYTKLSSSGT (SEQ ID NO: 49)). The main library was further expanded by the LoxP-cre system.

The selection of phage particles displaying specific scFv fragments was performed on Immuno 96MicroWell ^TM plates (Nunc, Denmark). First, 50 μg/ml of human ROR1 recombinant protein (Catalog No.: RO1-H522y, Acrobiosystems) in phosphate buffered saline (PBS) was coated on the plate overnight at 4°C. After blocking with 2% (w/v) milk powder in PBS (2% MPBS), a library containing about 10 ¹¹ phage particles was added and the plate was incubated at room temperature (RT; 25°C-28°C) for 2 hours. Unbound phages were eliminated by washing 10-20 times with PBS (PBS-T) containing 0.1% Tween 20, followed by washing 10-20 times with PBS. Bound phages were eluted by incubation with 50 μl of 1 μg/μl trypsin for 10 minutes, followed by incubation with 50 μl of 50 mM glycine hydrochloride (pH 2.0) (neutralized immediately after 10 minutes with 50 μl of 200 mM Na ₂ HPO ₄ (pH 7.5)). The eluted phages were used to infect exponentially growing E. coli TG1 cells by incubation at 37°C for 30 minutes. The infected cells were plated on TYE plates containing ampicillin (100 μg/mL) and glucose (1% w/v), which were then incubated overnight at 37°C. Single phage-infected colonies were picked and grown in 96-well plates to produce phagemid particles. The cultures were rescued using M13KO7 or KM13 helper phages. The rescued phage particles were used to initiate subsequent rounds of selection using similar conditions. Three rounds of selection were performed for the ROR1 protein.

To test ROR1 binding in enzyme-linked immunosorbent assay (ELISA), single clones from the last panning were selected and grown at 37°C and rescued with M13K07 helper phage. The amplified phage preparations were blocked for 1 hour with 5% skim milk in PBS at 37°C and added to 96-well microplates (Nunc) coated with ROR1 (Catalog No.: RO1-H522y, Acrobiosystems) (0.5 μg/ml). After incubation at 37°C for another 1 hour, the plates were washed 3 times with PBST and incubated with anti-M13 phage antibodies (Amersham) conjugated with mouse horseradish peroxidase (HRP). After careful washing, 3,30,5,50-tetramethylbenzidine (TMB, Sigma) was added as a substrate. The color reaction was measured at 450 nm using a Thermo multiskan ELISA reader (MA, USA).

In further testing, three clones were screened: ROR1-4, ROR1-12 and ROR1-14.

Example 2 Expression and purification of full-length antibodies

A method for producing full-length human IgG1 antibodies from scFv has been established. The genes encoding the VH and VL regions of anti-ROR1 antibodies are sequentially inserted into the expression vector pIgG containing the genes of the hIgG1 heavy chain constant region and the kappa light chain constant region. In order to express soluble antibodies in mammalian cells, recombinant pIgG is transiently transfected into human 293T cells using lipofectamine. The transfected cells are maintained in 293SFM at 37°C for 8 days. During this period, the culture medium is changed 2 times and the culture supernatant is collected. The full-length antibodies secreted into the culture medium are purified using protein A affinity chromatography (Pharmacia). The purified antibodies are concentrated to 1 mg/ml, sterile filtered, and characterized by SDS-PAGE, ELISA, and isothermal titration calorimetry (ITC).

Example 3 Physical and Chemical Analysis

Clones ROR1-4, ROR1-12 and ROR1-14 were further tested in size exclusion chromatography. Specifically, 20 μg of sample was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na ₂ SO ₄ (pH 7.0) as running buffer. The run time was 29 minutes. All measurements were performed on an Agilent 1220HPLC. Data were analyzed using OpenLAB software. The main peak of ROR1-12 was above 95% in SEC, indicating that the purified antibody had high purity and integrity.

Example 4 Anti-ROR1 Antibody Specific Binding to Human ROR1

An ELISA assay was used to determine the relative binding activity of the antibodies to recombinant human ROR1.

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 with 1% BSA in PBS for 1 hour at 37°C. 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 with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer for 1 hour at 37°C, washed again, developed with TMB and terminated with 1MH ₂ SO _4. The absorbance at 450nm-620nm was determined.

The EC ₅₀ and representative binding curves of the anti-ROR1 antibodies binding to human ROR1 are shown in FIG1 (VLS101 in FIG1 , below, and in the figures referred to therein refers to the ROR1 antibody in the VLS101 ADC), indicating that these clones specifically bind to human ROR1.

Example 5 Affinity of anti-ROR1 antibodies for human ROR1

5.1

Add buffer to the pre-wetted plate and pre-equilibrate the AHC sensor for 10 min. Before adding the sample, equilibrate the baseline with 1 X PBS, 0.02% Tween-20 for 100 s. ROR1 antibody-coupled AHC sensor: Dilute the ROR1 antibody to 5μg/mL with 1 X PBS, 0.02% Tween-20, and stop the solidification time at 200 s to prepare the ROR1 antibody-coupled sensor. Equilibrate the baseline with Running buffer (1 X PBS, 0.02% Tween-20) for 150 s. The analyte ROR1 Protein (ARCO, Catalog No. RO1-H522y) was diluted with Running buffer to 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM, and 1.56nM. The analyte binds for 200 s and dissociates for 600 s. Regenerate with regeneration solution for 30 s. The obtained data were fitted with a 1:1 binding model using Octet software and calculated using Global fitting.

Table 2 Affinity parameters of ROR1-4 and ROR1-14

5.2

The kinetic binding activity of anti-ROR1 antibodies to human ROR1 (Acro biosystems) was measured by surface plasmon resonance using the Biacore T200 system (Biacore, GE Healthcare).

Approximately 7000RU of goat anti-human Fcγ antibody (Jackson ImmunoReaserch, Cat. No. 109-005-098) was immobilized on a CM5 sensor chip by amino acid coupling chemistry. ROR1 antibody was bound to the surface of the immobilized goat anti-human IgG antibody. HBS-EP+ buffer was used as the running buffer. Different concentrations of human ROR1 protein (from 6.25nM to 200nM) were injected onto the antibody surface. After each cycle, the CM5 chip surface was regenerated by injecting 10mM glycine (pH1.5). The association rate Ka and dissociation rate Kd, as well as the equilibrium dissociation constant _KD , were analyzed by background subtraction combined with the sensorgram. The resulting data set was fitted with a 1:1 Langmuir binding model using the Biacore T200 evaluation software.

Table 3 Affinity parameters of ROR1-12 and ROR1-14

Example 6 Anti-ROR1 Antibody Has No Cross-Reaction with Mouse ROR1

ELISA assay was used to determine the relative binding activity of antibodies to mouse ROR1.

Mouse ROR1 protein (Catalog number: RO1-M5221, Acrobiosystems) was fixed to a 96-well plate by incubation overnight at 4°C. The plate was then blocked by incubation with 1% BSA in PBS for 1 hour at 37°C. 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 with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer for 1 hour at 37°C, washed again, developed with TMB and terminated with 1M H ₂ SO _4. The absorbance at 450nm-620nm was determined.

Representative binding curves for these antibodies are shown in Figure 2 , indicating that these clones did not bind to mouse ROR1.

Example 7 Cross-reaction experiment between anti-ROR1 antibody and human ROR2

ELISA assay was used to determine the relative binding activity of antibodies to human ROR2.

Human ROR2 (Catalog No.: RO2-H52E5, Acrobiosystems) was immobilized on a 96-well plate by incubation overnight at 4°C. Nonspecific binding sites were blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, the plate was washed 3 times with PBST (PBS containing 0.05% Tween20). Serial dilutions of anti-ROR1 antibodies and human IgG controls were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the immobilized protein at 37°C for 1 hour. After binding, the plate was washed 3 times with PBST, incubated with peroxidase-labeled donkey anti-human IgG (Jackson Immuno Research) diluted 1/15,000 in binding buffer at 37°C for 1 hour, washed again, developed with TMB and stopped with 1M H ₂ SO _4. The absorbance at 450nm-620nm was measured.

Representative binding curves of these antibodies are shown in Figure 3, indicating that ROR1-14 binds to human ROR2, but ROR1-12 does not bind to human ROR2.

Example 8 Preparation of Antibody Drug Conjugates ROR1-4-BL20MMAE and ROR1-12-BL20MMAE

The stock solutions of ROR1-4 and ROR1-12 antibodies targeting ROR1 were replaced with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH ₂ PO ₄ -Na ₂ HPO ₄ )/150 mM sodium chloride (NaCl)/2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4 reaction buffer to a concentration of 10 mg/mL, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added in a 10-fold excess molar ratio. The reaction solution was stirred at 28°C for 4 hours. The above reaction solution was cooled to 20°C, an appropriate amount of diethylacetamide (DMA) was added, and then a 5-fold excess molar ratio of compound Ic-4 (10 mg/ml pre-dissolved in DMA) was added to ensure that the volume of DMA in the reaction system did not exceed 10%, and the coupling was carried out at 25°C for 1.0 hour. The coupling reaction mixture was purified by gel filtration using a desalting column with sodium dihydrogen phosphate-disodium hydrogen phosphate/sucrose buffer at pH 7.4, and the peak samples were collected according to the UV280 ultraviolet absorption value. Then it was sterilized through a 0.22 micron pore size filter and stored at -80°C. The resulting antibody conjugates were named ROR1-4-BL20MMAE and ROR1-12-BL20MMAE.

The results are shown in Figures 4 to 9. The mass spectrometry of the humanized antibody ROR1-4 after deglycosylation (Figure 4) and the HIC and mass spectrometry after deglycosylation of its antibody conjugate ROR1-4-BL20MMAE (abbreviated as ROR1-4-BL20E) (Figures 5 and 6) all show that after the conjugation reaction, the antibody ROR1-4 forms the antibody conjugate ROR1-4-BL20MMAE, and the molecular weight of the conjugate is consistent with the expected value, with a DAR of approximately 4.0. The mass spectrometry deglycosylated spectrum of the humanized antibody ROR1-12 (Figure 7) and the HIC and mass spectrometry deglycosylated spectrum of its antibody conjugate ROR1-12-BL20MMAE (Figures 8 and 9) both indicate that after the conjugation reaction, the antibody ROR1-12 forms the antibody conjugate ROR1-12-BL20MMAE (abbreviated as ROR1-12-BL20E). The molecular weight of the conjugate is consistent with the expected value, and the DAR is approximately 4.0.

Example 9 Preparation of ROR1-12-GGFG-Dxd

The ROR1-12 antibody targeting ROR1 was replaced with a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH ₂ PO ₄ -Na ₂ HPO ₄ )/150mM sodium chloride (NaCl)/2mM ethylenediaminetetraacetic acid (EDTA), pH 7.0 reaction buffer to a concentration of 10 mg/mL, and a 10-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 28° C. for 4 hours. The above reaction solution was cooled to room temperature, an appropriate amount of diethylacetamide (DMA) was added, and then a 12-fold excess molar ratio of the compound GGFG-DXd (purchased from Shanghai Haoyuan Chemical, 10 mg/ml pre-dissolved in DMA) was added to ensure that the volume of DMA in the reaction system did not exceed 10%, and the coupling was carried out by stirring at 25° C. for 1.0 hour. The coupling reaction mixture was purified by gel filtration using a desalting column with a pH 6.6 4-morpholineethanesulfonic acid (MES)-Tris/sucrose buffer, and the peak sample was collected according to the UV280 ultraviolet absorption value. It was then sterilized through a 0.22 micron pore size filter and stored at -80°C. The resulting antibody conjugate was named ROR1-12-GGFG-Dxd.

The results are shown in Figures 10, 11, and 12. The mass spectrometry deglycosylation spectrum of the humanized antibody ROR1-12 (Figure 10) and the HIC and mass spectrometry deglycosylation spectrum of its antibody conjugate ROR1-12-GGFG-Dxd (abbreviated as ROR1-12-DX) (Figures 11 and 12) all indicate that after the conjugation reaction, the antibody ROR1-12 forms the antibody conjugate ROR1-12-DX, and the molecular weight of the conjugate is consistent with the expected value, with a DAR of approximately 8.0.

Example 10 In vivo antitumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human mantle cell lymphoma JeKo-1

The in vitro anti-tumor activity of ROR1 antibody-drug conjugates (ROR1-ADCs) against human mantle cell lymphoma JeKo-1, triple-negative breast cancer cells MDA-MB-231, and non-small cell lung cancer cells NCI-H1975 (all purchased from the Chinese Academy of Sciences Cell Bank) was determined by in vivo experiments, that is, allogeneic or xenografts of cancer cells were implanted in rodents and the tumors were treated with the combination. The test mice were treated with drugs or controls and monitored for several weeks or longer to measure the time to tumor doubling, logarithmic cell killing, and tumor inhibition.

The efficacy of ROR1-12-BL20E and ROR1-12-DX on subcutaneous transplanted tumors of JeKo-1 mice.

Each NOD-Scid mouse (Shanghai Lingchang Biotechnology Co., Ltd.) was subcutaneously inoculated with JeKo-1 cells. When the tumor grew to about 100 mm ³ , the mice were divided into groups according to the tumor volume and intravenously injected (IV) with drugs once every 4 days (Q4D) for a total of 2 times (D0, D4), with an injection volume of 10 mL/Kg; the dosage and administration schedule are shown in Table 4. The tumor diameter was measured with a vernier caliper twice a week, and the tumor volume (V) was calculated as: V = 1/2 × a × b ² (where a and b represent length and width, respectively).

Table 4

T/C (%) = (T-T0)/(C-C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Tumor growth inhibition rate % (TGI%) = 100-T/C (%).

When the tumor regresses, tumor growth inhibition rate % (TGI%) = 100-(T-T0)/T0×100

Unless otherwise specified, the tumor volumes or tumor weights between the two groups were compared using the two-tailed Student’s t test, and P < 0.05 was defined as a statistically significant difference.

ROR1-12-BL20E (5 mg/kg, IV, D0, 4) had a significant inhibitory effect on the growth of subcutaneous transplanted tumors of JeKo-1 mice with human mantle cell lymphoma, with a tumor inhibition rate of 92%, and 1/6 of the tumors partially regressed; ROR1-12-DX (5 mg/kg, IV, D0, 4) had an inhibition rate of 81% on subcutaneous transplanted tumors of JeKo-1 mice; tumor-bearing mice were well tolerated to the above drugs, and no obvious symptoms such as weight loss occurred. In comparison, the efficacy of ROR1-12-BL20E on JeKo-1 subcutaneous transplanted tumors was significantly stronger than that of ROR1-12-DX (P<0.05, comparison of equal dose groups), as shown in Figure 13 (Effects of ROR1-12-BL20E and ROR1-12-DX on the growth of subcutaneous transplanted tumors of JeKo-1 mice with human mantle cell lymphoma).

Example 11 Efficacy of ROR1 antibody-drug conjugates (ROR1-ADCs) ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on subcutaneous transplanted tumors of human breast cancer MDA-MB-231 in nude mice

The results showed that ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE (5 mg/kg, IV, QW×3) inhibited the growth of subcutaneous transplanted tumors of human breast cancer MDA-MB-231 mice, with the inhibition rates of 49%, 39%, and 24%, respectively (Figure 14, the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the growth of subcutaneous transplanted tumors of human breast cancer MDA-MB-231); tumor-bearing mice were able to tolerate the above drugs well, and no obvious symptoms such as weight loss occurred (Figure 15, the effects of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the weight of tumor-bearing mice). In comparison, ROR1-12-BL20E had a relatively good efficacy on subcutaneous transplanted tumors of MDA-MB-231 mice.

Example 12 Efficacy of ROR1 antibody-drug conjugates (ROR1-ADCs) ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on subcutaneous transplanted tumors of human lung cancer H1975 mice

Each BALB/c nude mouse (Beijing Huafukang Biotechnology Co., Ltd.) was subcutaneously inoculated with 5×10 ⁶ H1975 cells. When the tumor grew to 100-150 mm ³ , the mice were divided into groups according to the tumor volume and intravenously injected (IV) with drugs for 3 times with an injection volume of 10 mL/Kg. The tumor diameter was measured with a vernier caliper twice a week.

The results showed that ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE (5 mg/kg, IV, once a week, 3 times in total) had an inhibitory effect on the growth of subcutaneous transplanted tumors of human lung cancer H1975 mice, and the tumor growth inhibition rate (TGI%) was 69%, 43%, and 19%, respectively (Figure 16, the effect of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the growth of subcutaneous transplanted tumors of human lung cancer H1975); tumor-bearing mice were able to tolerate the above drugs well, and no obvious symptoms such as weight loss occurred (Figure 17, the effect of ROR1-12-BL20E, ROR1-4-BL20E, and VLS101-MMAE on the body weight of tumor-bearing mice). In comparison, ROR1-12-BL20E had the best efficacy on subcutaneous transplanted tumors of H1975 mice (P>0.05, comparison of equal dose groups).

The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown herein and discussed in the specific embodiments, 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 (25)

An antibody or an antigen-binding fragment thereof targeting ROR1, comprising: A heavy chain variable region, the heavy chain variable region comprising: a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 17 and 33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 19 and 35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 5, 21 and 37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and A light chain variable region, the light chain variable region comprising: d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 7, 23 and 39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; e) a complementary determining region VL CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 9, 25 and 41, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity thereto; and f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 11, 27 and 43, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) a heavy chain variable region, the heavy chain variable region comprising: a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and c) a complementarity determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and A light chain variable region, the light chain variable region comprising: d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or (2) a heavy chain variable region, the heavy chain variable region comprising: a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; b) a complementary determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and A light chain variable region, the light chain variable region comprising: d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or (3) a heavy chain variable region, the heavy chain variable region comprising: a) a complementary determining region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; b) a complementarity determining region VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and c) a complementary determining region VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and A light chain variable region, the light chain variable region comprising: d) a complementary determining region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; e) a complementary determining region VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and f) a complementary determining region VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto. The antibody or antigen-binding fragment thereof according to claim 2, comprising: (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:14, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or (3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:46 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto. The antibody or antigen-binding fragment thereof according to claim 3, comprising: (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14; (2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30; or (3) a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises: (1) the heavy chain amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and the light chain amino acid sequence of SEQ ID NO:16, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; (2) the heavy chain amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and the light chain amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; or (3) the heavy chain amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto; and the light chain amino acid sequence shown in SEQ ID NO:48, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical thereto. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises: (1) the heavy chain amino acid sequence shown in SEQ ID NO:15 and the light chain amino acid sequence shown in SEQ ID NO:16; (2) the heavy chain amino acid sequence shown in SEQ ID NO:31 and the light chain amino acid sequence shown in SEQ ID NO:32; or (3) The heavy chain amino acid sequence shown in SEQ ID NO:47 and the light chain amino acid sequence shown in SEQ ID NO:48. The antibody or antigen-binding fragment thereof according to 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 chimeric antibody, a single-chain antibody, Fv, a single-chain Fv (scFv), Fd, Fab, Fab' and F(ab')2. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which binds to human ROR1 with a KD of 2.0×10 ^-9 M or less. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which does not cross-react with mouse ROR1. A nucleic acid sequence encoding an antibody or an antibody thereof according to any one of claims 1 to 9 Original binding fragment. A plasmid or vector comprising the nucleic acid sequence according to claim 10. A host cell comprising and expressing the nucleic acid sequence according to claim 10 or the plasmid or vector according to claim 11. An antibody-drug conjugate having a structure shown in Formula I: Ab-(LD) _n (Formula I), wherein Ab is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9; L is a linker; D is a cytotoxic drug; and n is a number between 1-10, preferably a value between 1-8; preferably n is 1, 2, 3, 4, 5, 6, 7, 8, and any value between any two values. The antibody-drug conjugate according to claim 13, wherein L is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linker, disubstituted maleimide, and includes one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG). The antibody-drug conjugate according to claim 13, wherein D is selected from one or more of the following groups: (i) Tubulin inhibitors, such as maytansine derivative DM1, maytansine derivative DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF); (ii) Toxins targeting DNA, such as duocarmycin and pyrrolobenzodiazepine (PBD); (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd. A composition comprising the antibody-drug conjugate according to any one of claims 13 to 15. Use of the antibody-drug conjugate according to any one of claims 13 to 15 or the composition according to claim 16 in the preparation of a medicament for treating cancer. A method for treating cancer, comprising administering the antibody-drug conjugate according to any one of claims 13-15 or the composition according to claim 16 to a subject in need thereof. The method of claim 18, wherein the cancer is a hematological cancer or a solid tumor. The method of claim 18 or 19, wherein the cancer is selected from one or more of the following: lymphoma, CLL, small lymphocytic lymphoma, marginal cell B-cell lymphoma, Burkett's lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer and head and neck cancer. The method according to any one of claims 18 to 20, wherein the antibody-drug conjugate or the composition is administered as a single active pharmaceutical ingredient or in combination with other anticancer drugs. The method according to claim 21, wherein the other anti-cancer drugs are selected from one or more of the following: anti-PD-1 antibody, anti-LAG-3 antibody, anti-CTLA-4 antibody, IL-2, IL-21, anti-CD137 antibody and anti-GITR antibody. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a kit for diagnosing a disease. A kit for diagnosing a disease, the kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and instructions for use. A method for diagnosing a disease, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the kit according to claim 24.