Antibody for anti-ROR1 protein and conjugate thereof

Abstract

The present invention relates to an anti-ROR1 antibody, an antibody-drug conjugate comprising the antibody, a pharmaceutical composition and a medical product comprising the antibody-drug conjugate, and a preparation method and use of the antibody-drug conjugate, the antibody-drug conjugate being effective for treating and / or preventing ROR1 positive tumors, including hematoma and solid tumors, such as tumor cells, tumor cells, tumor cells, and the like. Cancers such as breast cancer and bladder cancer.

Description

An antibody against ROR1 protein and its conjugate Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to an anti-ROR1 protein antibody, a conjugate, and their use in treating or preventing tumors. Background Art

[0002] ROR1, short for receptor-tyrosine-kinase-like orphan receptor 1, is a type I single-pass transmembrane protein and a member of the receptor tyrosine kinase (RTK) family. It is lowly expressed during embryonic development but highly expressed in a variety of malignant tumors and tissues, such as chronic lymphocytic leukemia (CLL), allergic lymphocytic leukemia (ALL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma. Numerous data indicate that ROR1 plays a significant role in promoting tumor growth and metastasis, inducing tumor cell drug resistance, and inhibiting apoptosis. The human ROR1 molecule consists of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region includes an immunoglobulin-like domain (Ig-like), a cysteine-rich domain or frizzled domain (CRD or FZD), and a Kring (KNG) domain (KRD); the intracellular region contains a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / ThrD), and a proline-rich domain (PRD).

[0003] Studies have shown that the currently known physiological functions of ROR1 are: (1) It plays an important role in mouse growth: it is expressed in the face, limbs, heart, and lungs of mice; although ROR1-deficient mice can survive, they show respiratory disorders and die within 24 hours after birth; (2) During embryonic development, ROR1 plays a key role in regulating the development of embryonic muscles and bones, including regulating cell division, proliferation, migration, etc., and participating in the generation of organs such as nerves, bones, and blood vessels.

[0004] During cancer progression, overexpression of ROR1 can promote tumor growth. ROR1 can bind to Wnt5a, participate in Wnt signal transduction, and interact with signaling pathways such as EGFR and Met to promote the growth, proliferation, and migration of tumor cells. ROR1 and CK1ε (casein kinase 1) activate AKT / PI3K, thereby activating the pCREB pathway, leading to cell proliferation; ROR1 enhances EGFR signaling and induces epithelial-mesenchymal transition (EMT); ROR1 is highly expressed in most hematological tumors and solid tumors. For example, the proportion of ROR1+ in CLL patients exceeds 90%; in some solid tumors, such as lung cancer, pancreatic cancer, and colorectal cancer cells, ROR1 expression is also significantly increased and is closely related to disease progression and treatment efficacy. Therefore, ROR1 can be used as a specific targeted marker for the development of tumor treatment drugs.

[0005] Breast cancer remains the most common female cancer worldwide, with the global incidence rate increasing by 0.2% to 8% annually. Approximately 1.4 million people are diagnosed with breast cancer worldwide each year, and approximately 500,000 die from the disease. Breast cancer is the leading cause of death among women aged 40 to 55. Since the late 1970s, breast cancer has consistently ranked as the leading malignant tumor among women worldwide. According to the American Cancer Society, there are approximately 200,000 new cases of breast cancer in the United States each year, with an incidence rate of 116 per 100,000 women. For American women, the lifetime risk of developing breast cancer by age 85 is approximately 12.5%, meaning one in every 8-10 women will develop breast cancer, and the lifetime risk of dying from it is approximately 3.4%. However, with advances in medical care and the continuous development of new drugs, while the global incidence of breast cancer has increased by 3% annually over the past decade, the survival rate for breast cancer patients has increased by 20%. Although my country has a low incidence of breast cancer in women worldwide, the incidence rate has increased significantly in recent years. Over the past decade, breast cancer incidence rates in major Chinese cities have increased by 37%, while mortality rates have risen by 38.9%. In rural areas, the mortality rate has increased by 39.7%. Of concern is that the average annual growth rate of breast cancer in my country is 1-2 percentage points higher than in high-incidence countries, increasing at a rate of 3-4% per year, making it the fastest-growing cancer mortality rate in cities. The age of onset is also trending younger. Shanghai, Beijing, Tianjin, and coastal areas are particularly high-incidence areas for breast cancer, where it now ranks first among female malignant tumors.

[0006] Endometrial cancer, also known as uterine corpus cancer, is one of the three most common malignant tumors of the female reproductive tract, primarily occurring in perimenopausal and postmenopausal women. With increasing life expectancy and changing lifestyles, the incidence of endometrial cancer has been steadily increasing over the past two decades, with a younger onset. In Western countries, endometrial cancer ranks first among female reproductive tract malignancies. In my country, according to the "2015 Analysis of the Epidemiology of Malignant Tumors in China" published by the National Cancer Center in 2019, endometrial cancer resulted in approximately 69,000 cases and 16,000 deaths in 2015, with an incidence rate of 10.28 per 100,000 people, accounting for 3.88% of all female malignant tumors. It is the second most common gynecological malignancy after cervical cancer, accounting for approximately 20% to 30% of all gynecological malignancies. In some developed cities, endometrial cancer has become the leading gynecological malignancy.

[0007] Prostate cancer is the most prevalent cancer in men, accounting for 13% of all cancer cases. It accounts for 7% of all cancer-related deaths, making it the second most common cause of cancer death in men. The age-standardized incidence of prostate cancer has increased 41% since 1993, even though the mortality rate has decreased 10% over the past decade. It is a heterogeneous disease with a wide range of pathogenesis, from asymptomatic and long-term men dying from the disease to serious malignancies in men who die from the disease or have significant morbidity.

[0008] Currently, there are four ADC drugs targeting ROR1, one humanized monoclonal antibody drug, five bispecific antibody drugs, and four CAR-T drugs. The fastest clinical progress is Phase III, and no products have been launched on the market. Among the ADC drugs currently in the clinical stage, VLS101 is in Phase III clinical trials. VelosBio announced the Phase I clinical data of VLS101 for the treatment of lymphoma in October 2021. The results showed that it achieved good clinical results. However, due to its non-site-specific coupling method, the shedding of small molecules caused by the reverse Michael addition reaction caused blood toxicity, posing a drug safety risk. The market needs more therapeutic drugs targeting the ROR1 target to meet the diverse treatment needs of patients.

[0009] Summary of the Invention

[0010] In a first aspect, the present application provides a conjugate comprising an anti-ROR1 antibody or an antigen-binding fragment of the antibody coupled to one or more drug molecules.

[0011] In a second aspect, the present application provides a pharmaceutical composition comprising the conjugate described in the first aspect and a pharmaceutically acceptable carrier.

[0012] In a third aspect, the present application provides use of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a medicament for treating or preventing cancer.

[0013] In a fourth aspect, the present application provides a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the conjugate of the first aspect or the pharmaceutical composition of the second aspect to the individual suffering from the cancer.

[0014] In a fifth aspect, the present application provides a medical product (eg, a medicine kit) comprising the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect.

[0015] In a sixth aspect, the present application provides use of the conjugate described in the first aspect and an anti-proliferative agent in the preparation of a drug for treating tumors.

[0016] In a seventh aspect, the present application provides a pharmaceutical composition comprising the conjugate according to the first aspect and an anti-proliferative agent.

[0017] In an eighth aspect, the present application provides a method for treating a tumor in an individual, comprising administering a therapeutically effective amount of the conjugate of the first aspect or the pharmaceutical composition of the second aspect and an anti-proliferative agent to the individual having the tumor.

[0018] In a ninth aspect, the present application provides an anti-ROR1 antibody or an antigen-binding fragment of the antibody, a pharmaceutical composition comprising the antibody or antigen-binding fragment, pharmaceutical uses of the antibody or antigen-binding fragment, and a method for treating tumors / cancer using the antibody or antigen-binding fragment.

[0019] In one embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

[0020] 1) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NOs. 1, 2 and 3, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NOs. 4, 5 and 6, respectively; or

[0021] 2) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NOs. 1, 9 and 3, respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NOs. 4, 5 and 6, respectively; or

[0022] 3) The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NOs. 1, 11 and 3, respectively; the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NOs. 4, 5 and 6, respectively.

[0023] In the aforementioned embodiments, the antibody may be a monoclonal antibody, a bispecific antibody, or a humanized antibody.

[0024] In the aforementioned embodiment, the antibody may be an IgG type antibody, preferably an IgG1 type antibody.

[0025] In the aforementioned embodiment, the antigen-binding fragment may be a Fab fragment, a F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0026] In the aforementioned embodiment, the amino acid sequence of the heavy chain variable region may be selected from SEQ ID NO.7, SEQ ID NO.10 and SEQ ID NO.12, and / or the amino acid sequence of the light chain variable region may be SEQ ID NO.8.

[0027] In the aforementioned embodiment, the heavy chain of the antibody may comprise a heavy chain constant region of the amino acid sequence shown in SEQ ID NO.13, and / or the light chain of the antibody may comprise a light chain constant region of the amino acid sequence shown in SEQ ID No.19.

[0028] In the aforementioned embodiment, the C-terminus of the light chain of the antibody or antigen-binding fragment thereof is linked to a glutamine-containing tag peptide, and the glutamine-containing tag peptide is selected from LQSGA, GGLQSGA, and GGGLQSGA.

[0029] The present invention provides a conjugate comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of the preceding embodiments coupled to one or more drug molecules, wherein the structure of the conjugate is Ab-(LU)n, wherein Ab represents the anti-ROR1 antibody or antigen-binding fragment thereof, L represents a linker, U represents a drug molecule, and n is an integer or decimal from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8; n is preferably 4.

[0030] In the aforementioned embodiments, the drug molecule may be an anticancer drug, for example, a cytotoxic drug, an immunopotentiator, or a radioisotope.

[0031] In the aforementioned embodiments, the cytotoxic drug may be selected from a tubulin inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, an antimetabolite, or an antitumor antibiotic.

[0032] In the aforementioned embodiment, the tubulin inhibitor may be selected from auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine, or dolastatin and its derivatives);

[0033] The DNA topoisomerase inhibitor can be selected from camptothecin analogs or DNA topoisomerase I inhibitors and derivatives thereof, for example, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyecliptin, topotecan, lertotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4- dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide;

[0034] The DNA damaging agent can be selected from calicheamicin, duocarmycin, and anthramycin derivative PBD (pyrrolobenzodiazepine);

[0035] The antimetabolite may be selected from methotrexate, 6-mercaptopurine or 5-fluorouracil; and / or

[0036] The anti-tumor antibiotic can be selected from polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride).

[0037] In the aforementioned embodiment, the immunopotentiator may be selected from levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic-cytidylic acid, or polyinosinic-uridylic acid.

[0038] In the aforementioned embodiment, the radioisotope may be selected from 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 60 Co or 177 Lu.

[0039] In the aforementioned embodiment, the drug molecule and the antibody or antigen-binding fragment thereof can be coupled via a linker, and the linker and the antibody or antigen-binding fragment thereof can be connected via a sulfhydryl group or an amino group.

[0040] In the aforementioned embodiments, the linker can be independently selected from mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG) m -Val-Cit, NH2-(PEG) m -Val-Cit-pAB and NH2-(PEG) m -Val-Cit-pABC, wherein m in (PEG)m is an integer from 1 to 8; preferably, m is 3.

[0041] In the aforementioned embodiment, the C-terminus of the light chain of the antibody or antigen-binding fragment thereof may be connected to a glutamine-containing tag peptide, wherein the glutamine-containing tag peptide is selected from LQSGA, GGLQSGA and GGGLQSGA, and the linker is connected to the side chain of the glutamine residue in the glutamine-containing tag peptide through an amide bond.

[0042] In the aforementioned embodiment, the antibody may comprise Q295 in the heavy chain constant region thereof, and the linker is connected to the side chain of Q295 via an amide bond.

[0043] In some embodiments, the linker is connected to the side chain of the glutamine residue in the glutamine-containing tag peptide through an amide bond.

[0044] The present invention also provides a pharmaceutical composition comprising: the conjugate according to any one of the aforementioned embodiments, a pharmaceutically acceptable carrier, and optionally an anti-proliferative agent.

[0045] In the aforementioned embodiments, the anti-proliferative agent may be selected from paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, or iproplatin.

[0046] The present invention also provides use of the conjugate or pharmaceutical composition according to any one of the aforementioned embodiments in the preparation of a medicament for treating or preventing cancer.

[0047] The present invention also provides a method of treating cancer in an individual, comprising administering a therapeutically effective amount of the conjugate or pharmaceutical composition of any one of the preceding embodiments to the individual suffering from the cancer.

[0048] In some embodiments, the cancer is a solid tumor, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, or gallbladder cancer, preferably adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung, or ovary; more preferably gastric cancer or pancreatic cancer; in some embodiments, the cancer is a blood tumor, preferably mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or Richter's transformed lymphoma.

[0049] In the aforementioned embodiments, the cancer may be a ROR1-positive cancer.

[0050] The present invention also provides a medical product comprising the conjugate or pharmaceutical composition according to any one of the aforementioned embodiments; the medical product may be in the form of a medicine kit, which comprises a container containing the conjugate or the pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG1 : Schematic diagram of the structure of the conjugate Hu17-H2L1 / H3L1 / H4L1-4LND1002 ADC according to an exemplary embodiment of the present invention.

[0052] The left side of the figure shows the structure of the antibody, and the right side shows the corresponding linker-drug structure. Each molecule consists of an anti-ROR1 humanized monoclonal antibody (Hu17-H2L1 / H3L1 / H4L1) coupled to one molecule of MMAE or MMAE derivative at the Q295 (EU numbering) amino acid of each heavy chain through a linker. At the same time, a glutamine-containing tag peptide (GGLQSGA) is connected to the C-terminus (carboxyl terminus) of each light chain, which is coupled to one molecule of MMAE derivative through a linker. The connection between the antibody and the linker is a stable amide bond (isopeptide bond), and the average drug molecule to antibody ratio (DAR) is 4.0.

[0053] The right side of the figure shows the structure of LND1002 before conjugation, which includes a drug molecule, such as MMAE or an MMAE derivative, and a linker, NH2-PEG3-Val-Cit-pABC. After conjugation, the terminal NH2 group of the linker is connected to the side chain (-CH2-CH2-C(=O)-NH2) of the glutamine residue (Q) in the heavy chain Q295 or the glutamine-containing tag peptide at the end of the light chain to form a "-CH2-CH2-C(=O)-NH-PEG3-Val-Cit-pABC" structure.

[0054] Figure 2: Affinity of murine and humanized antibodies to MDA-MB-231-ROR1 at the cellular level.

[0055] Figure 3: UC961-VC MMAE DAR detection spectrum.

[0056] Figure 4: Internalization assay of various ADCs in MDA-MB-231-ROR1 cells.

[0057] Figure 5: Inhibitory effect curves of various ADCs on human lung adenocarcinoma H1975 mouse transplanted tumors.

[0058] Figure 6: Inhibitory effect curves of various ADCs on human breast cancer HCC1187 mouse transplanted tumors

[0059] Figure 7: Dose-time curves of two ADCs after three doses in cynomolgus monkeys.

[0060] Figure 8: Free small molecule concentration-time curves of two ADCs after three doses in cynomolgus monkeys. Specific implementation plan

[0061] definition

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0063] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" recorded should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges terminating with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0064] As used herein, the terms "pharmaceutical composition", "combination drug" and "drug combination" are used interchangeably and refer to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as they can work together to achieve the purpose of the present application. For example, the ingredients contained in the pharmaceutical composition (such as antibodies, nucleic acid molecules, nucleic acid molecule combinations and / or conjugates according to the present application) can be administered to the subject as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered separately to the subject, the ingredients can be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the application is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.The composition according to the application may contain wetting agents, emulsifiers or buffer substances as additives.

[0065] The pharmaceutical composition, vaccine or pharmaceutical preparation according to the present application can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

[0066] As used herein, "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the condition being treated.

[0067] As used herein, the term "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0068] The term "antibody" broadly encompasses intact antibodies and any antigen-binding fragment ("antigen-binding portion") or single-chain form thereof. A "full-length / intact antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region, which comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region, which comprises one domain: CL. The VH and VL regions are further subdivided into highly variable regions known as complementarity determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. Chimeric or humanized antibodies are also encompassed in antibodies according to the present application. Full-length / intact antibodies can be antibodies of any type, such as IgD, IgE, IgG, IgA, or IgM (or the subclasses described above), but antibodies do not need to belong to any specific class. According to the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be designated as different classes. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0069] Complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are a variety of common definitions for the CDR sequences of VH or VL, including Kabat, IMGT, Chothia, and Contact definitions. For the variable region sequences of a given antibody, the CDR sequences in the VH and VL sequences can be determined according to the Kabat, IMGT, Chothia, or Contact definitions. In this application, unless otherwise expressly stated, the amino acid sequences of the CDRs described herein (e.g., SEQ ID No. 1-6, 9, and 11) are all shown in accordance with the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in this area by a variety of methods. Although the scope of protection claimed in the claims of this application is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of this application.

[0070] The term "humanized antibody" refers to an antibody that may contain CDR regions derived from human antibodies and the rest of the antibody molecule is derived from one (or several) human antibodies. In addition, some residues in the framework (called FR) segment may be modified to retain binding affinity, and humanized antibodies or fragments thereof according to the present application may be prepared by techniques known to those skilled in the art.

[0071] The term "semi-humanized antibody" refers to an antibody in which one antibody chain comprises a murine variable region (as in a chimeric antibody) and the other antibody chain comprises a humanized variable region, as opposed to a humanized antibody or a fully humanized antibody.

[0072] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody. The chimeric antibody or its fragment according to the present application can be prepared by using genetic recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA, which contains a promoter and a sequence encoding the variable region of a non-human, especially mouse, monoclonal antibody according to the present application, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present application encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from the mouse DNA, and its isotype being determined by the constant region derived from the human DNA. For methods of preparing chimeric antibodies, for example, reference can be made to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

[0073] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0074] The term "bispecific antibody" refers to an antibody that has the ability to bind to two antigenic epitopes simultaneously. The two antigenic epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two binding parts fused to them (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding part can be a single-chain antibody (scFv) or a Fab fragment.

[0075] As used herein, the term "antigen-binding fragment" refers in particular to antibody fragments such as Fv, scFv (sc refers to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments or bispecific (diabody), or any fragment that can increase half-life by chemical modification, such as the addition of poly (alkylene) glycols such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes. The antigen-binding fragments of the anti-ROR1 antibodies of the present application have ROR1 binding activity. For example, the antigen-binding fragment is composed of or contains a partial sequence of the heavy chain or light chain variable chain of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. Such an antigen-binding fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

[0076] Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv)2, which can comprise two VH domains connected by a peptide linker and two VL domains, the two VL domains being combined with the two VH domains via a disulfide bridge.

[0077] The terms "Fc fragment", "Fc domain", "Fc part" or similar terms refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 segment and the CH3 segment of the constant region.

[0078] Generally, to prepare monoclonal antibodies or antigen-binding fragments thereof, especially murine monoclonal antibodies or antigen-binding fragments thereof, reference can be made to the techniques described in, inter alia, the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0079] The term "homology / identity / identity" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for alignment are well known in the art.

[0080] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0081] The term "ADC" refers to an antibody-drug conjugate, in which a small molecule drug with activity is chemically linked to an antibody or its antigen-binding fragment via a linker. Herein, the terms "ADC," "antibody-drug conjugate," and "conjugate" are used interchangeably.

[0082] As used herein, the term "cancer" refers to a proliferative disorder caused by or characterized by the proliferation of cells that have lost their susceptibility to normal growth control. The term "cancer" includes tumors and any other proliferative disorder. Cancers of the same tissue type originate from the same tissue and can be divided into different subtypes based on their biological characteristics.

[0083] "UC961-vc-MMAE," also known as VLS-101, is an ADC derived from the conjugation of the tubulin polymerization inhibitor MMAE to the cysteine ​​residue of the antibody Cirmtuzumab (UC961) using the cleavable linker MC-Val-Cit-PAB. The average DAR is 4. The antibody sequence can be found in sequences 3-6 of CN111587124A.

[0084] As used herein, the terms "NH2-PEG3-Val-Cit" and "NH2-PEG3-Val-Cit-pABC" have the same meaning and refer to the following structure:

[0085] The * position is connected to the bioactive molecule X, such as MMAE. When it is connected to the antibody, it has the following structure:

[0086] The amino group (-NH-) is used to connect the antibody.

[0087] The term "MMAE" refers to monomethyl auristatin E, which has the following structure:

[0088] When connected to the connector, it has the following structure:

[0089] ROR1 antibodies and conjugates of the present invention

[0090] In response to the deficiencies in the prior art, the inventors have developed an anti-ROR1 humanized antibody that can bind to ROR1 on ROR1-positive cells and internalize efficiently, making it very suitable for the development of ADCs. The drug molecule, such as MMAE, is coupled to the aforementioned anti-ROR1 antibody via the mTgase enzyme to obtain an antibody-drug conjugate with excellent uniformity of DAR=4. In subsequent ADC research and development, the inventors of the present application found that the use of the linker of the present application achieved good stability in the conjugation of the linker and the drug molecule, thereby coupling the humanized antibody to a small molecule drug (e.g., MMAE) via a linker. The resulting ADC drug has a strong killing effect on ROR1-high-expressing cancer cells, especially pancreatic cancer, gastric cancer, and lung cancer cells, and has excellent stability. In addition, the ADC of the present invention adopts a unique design, adding a glutamine-containing GGLQSGA peptide segment at the end of the light chain to achieve a higher drug loading of DAR=4, improving the therapeutic efficacy of the drug without significantly increasing toxicity. In vivo experiments showed that intravenous administration of the antibody-drug conjugate in nude mice with ROR1-positive gastric or pancreatic xenograft tumors resulted in dose-dependent tumor growth inhibition, with significant overall therapeutic effect. The ADC drug obtained in this application can produce a bystander effect, further enhancing the therapeutic effect. At the same dose, the in vivo efficacy is better than that of VLS101. In pre-toxicological studies in cynomolgus monkeys, the 9 mg / kg dose had similar hematologic toxicity to that of VLS101 6 mg / kg, greatly improving the therapeutic window.

[0091] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that can specifically bind to ROR1. Specifically, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto; and / or (ii) the light chain variable region comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has an amino acid sequence as shown in SEQ ID NO: 4, 5 or 6, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively; and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 1, 9 and 3, respectively; and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 1, 11 and 3, respectively; and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: GFSLSTSGMG (SEQ ID No.20), IWWDDDK (SEQ ID No.21), ARPQFITTVVAFYWYFDV (SEQ ID No.22); and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: ENIYSN (SEQ ID No.23), AAT, QHFWGTPWT (SEQ ID No.6), using the IMGT encoding method.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: GFSLSTSGM (SEQ ID No.24), WWDDD (SEQ ID No.25), PQFITTVVAFYWYFDV (SEQ ID No.3); and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: RASENIYSNLA (SEQ ID No.4), AATNLAD (SEQ ID No.5), QHFWGTPWT (SEQ ID No.6), using the Chothia encoding method.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: STSGMGVA (SEQ ID No.26), WLAHIWWDDDKR (SEQ ID No.27), ARPQFITTVVAFYWYFD (SEQ ID No.28); and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions respectively have the following amino acid sequences: YSNLAWY (SEQ ID No.29), LLVYAATNLA (SEQ ID No.30), QHFWGTPW (SEQ ID No.31), using Contact coding.

[0098] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application are isolated.

[0099] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0100] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 10, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

[0101] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

[0102] In some embodiments, the antibodies of the present application are monoclonal antibodies.

[0103] In some embodiments, the antibodies of the present application are bispecific antibodies. For example, one arm of the bispecific antibody can be an antigen-binding fragment (e.g., Fab or scFv) of the anti-ROR1 antibody described herein, and the other arm can be an antigen-binding fragment (e.g., Fab or scFv) targeting other antigens (e.g., other antigen targets that can be used for ADC construction) or targeting another ROR1 antigen epitope (different from the ROR1 binding epitope of the anti-ROR1 antibody described herein).

[0104] In some embodiments, the antibodies of the present application are humanized antibodies, including semi-humanized antibodies.

[0105] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application have ADCC activity.

[0106] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application have CDC activity.

[0107] In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of IgG1 subtype, IgG2 subtype, and IgG4 subtype.

[0108] In some embodiments, the heavy chain constant region of the antibody can be of human IgG1 subtype, human IgG2 subtype, human IgG4 subtype, murine IgG1 subtype, or murine IgG2a subtype.

[0109] In some embodiments, the heavy chain constant region is of the IgG1 subtype, ie, the antibody is an IgG1 type antibody.

[0110] In some embodiments, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

[0111] In some embodiments, the light chain constant region of the antibody can be human κ subtype, human λ subtype, murine κ subtype, or murine λ subtype.

[0112] In some embodiments, the antibody has a human IgG1 heavy chain constant region and contains a mutation at one or more of L234, L235, P329, and P331, wherein the residues are numbered according to the EU numbering system, for example, the mutation is one or more of L234A, L235A, P329A, and P331S.

[0113] In some embodiments, the antibody has a human IgG1 heavy chain constant region and contains the quadruple mutations L234A, L235A, P329A, and P331S.

[0114] In some embodiments, the antibodies of the present application are IgG1κ antibodies.

[0115] In some embodiments, the heavy chain of the antibody comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO. 13, and / or the light chain of the antibody comprises a light chain constant region with an amino acid sequence as shown in SEQ ID No. 19. In some embodiments, the heavy chain of the antibody comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO. 13, and / or the light chain of the antibody comprises a light chain constant region with an amino acid sequence as shown in SEQ ID No. 14.

[0116] In some embodiments, the antibodies or antigen-binding fragments thereof of the present application can be used to treat or prevent cancer, wherein the cancer overexpresses ROR1.

[0117] In one embodiment, the antibody capable of binding to ROR1 binds to a native epitope of ROR1 present on the surface of living cells. In one embodiment, the antibody capable of binding to ROR1 binds to the extracellular domain of ROR1. In one embodiment, the antibody capable of binding to ROR1 binds to the extracellular region of ROR1.

[0118] In some embodiments, a full-length antibody is provided, wherein a tag peptide containing glutamine is further linked to the C-terminus (carboxyl terminus) of the light chain of the antibody.

[0119] In some embodiments, the tag peptide containing glutamine includes: LQSGA, GGLQSGA, GGGLQSGA.

[0120] In another aspect, the present application provides an isolated polynucleotide encoding the antibody of the present application.

[0121] In another aspect, the present application provides a combination of isolated polynucleotides, comprising a polynucleotide encoding the light chain of the antibody or antigen-binding fragment thereof of the present application and a polynucleotide encoding the heavy chain of the antibody or antigen-binding fragment thereof of the present application.

[0122] In another aspect, the present application provides an expression vector comprising a polynucleotide described herein or a combination of polynucleotides described herein, wherein the polynucleotide is operably linked to a regulatory sequence that allows the encoded polypeptide to be expressed in a host cell or a cell-free expression system.

[0123] In some embodiments of the present application, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic host cell may be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as S. frugiperda, a plant cell such as tobacco, or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell described herein is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0124] In another aspect, the present application provides an antibody-drug conjugate comprising the anti-ROR1 antibody or antigen-binding fragment thereof of the present application coupled to one or more drug molecules. For descriptions of the embodiments and technical features of the anti-ROR1 antibody or antigen-binding fragment thereof of the present application, see above.

[0125] Given that ROR1 is primarily a molecular target of cancer / tumor cells, in some embodiments, the drug molecule is an anticancer drug. However, those skilled in the art will appreciate that when ROR1 is a target for other diseases other than cancer / tumor cells, the drug molecule can be selected based on the target disease.

[0126] Types of anticancer drugs include, but are not limited to, cytotoxic drugs, immunopotentiators, or radioisotopes.

[0127] In some embodiments, types of cytotoxic drugs include tubulin inhibitors (eg, alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.

[0128] In some embodiments, the cytotoxic drug moiety can be selected from the group consisting of, for example, anti-tubulin agents, DNA alkylating agents, DNA cross-linking agents, DNA intercalating agents, and RNA polymerase II inhibitors. In some embodiments, the cytotoxic drug moiety is selected from the group consisting of monomethyl auristatin E (MMAE), azonafide, α-amanitin, duocarmycin™, pyrrolobenzodiazepine (PBD), anthracycline neomycin metabolite PNU-159682 (CAS No: 202350-68-3), and pharmaceutically acceptable salts, esters, and analogs thereof.

[0129] In some embodiments, tubulin inhibitors include, but are not limited to, auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine, or dolastatin and its derivatives).

[0130] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and its derivatives, for example, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, lertotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3, 4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0131] In some embodiments, DNA damaging agents include, but are not limited to, calicheamicins, duocarmycins, and anthramycin derivatives such as PBD (pyrrolobenzodiazepine).

[0132] In some embodiments, immune enhancers include, but are not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic-cytidylic acid, or polyinosinic-uridylic acid.

[0133] In some embodiments, antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, or 5-fluorouracil.

[0134] In some embodiments, antitumor antibiotics include, but are not limited to, polypeptide antibiotics (eg, actinomycin D or bleomycin) or anthraquinone drugs (eg, doxorubicin or mitoxantrone hydrochloride).

[0135] In some embodiments, radioisotopes include, but are not limited to, 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 60 Co or 177 Lu.

[0136] In some embodiments, an antibody having the ability to bind to ROR1 is covalently linked to a drug moiety via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker can be cleaved under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH of less than 5.5. In some embodiments, the linker can be cleaved by intracellular proteases. In some embodiments, the linker is a cathepsin cleavable linker. In some embodiments, the linker comprises a dipeptide. In some embodiments, the dipeptide is valine (Val)-citrulline (Cit). In some embodiments, the antibody is connected to the linker via the cysteine ​​sulfhydryl group of the antibody. In one embodiment, the antibody is connected to the linker via the amino group of the antibody (particularly the amino group of the glutamine residue).

[0137] Non-limiting examples of linkers include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG) m -Val-Cit, NH2-(PEG) m -Val-Cit-pAB and NH2-(PEG) m -Val-Cit-pABC, wherein m is an integer from 1 to 8.

[0138] In some embodiments, the antibody-drug conjugates described herein have the following general formula Ab-(LU)n, wherein Ab represents the antibody targeting ROR1 of the present application, and L is a linker (e.g., NH2-(PEG) m -Val-Cit, NH2-(PEG) m -Val-Cit-pAB, NH2-(PEG) m -Val-Cit-pABC, mc-Val-Cit-pAB or Val-Cit, wherein m represents the number of PEG, which can be an integer from 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7 or 8), U is a drug (for example, DM1, DM4, MMAE, MMAF, DXD and SN38), and n represents the drug-antibody ratio (DAR). The DAR value can be an average value or any value from 1 to 8 (not limited to an integer, it can also be a decimal), preferably an integer from 1 to 8 (for example, 1, 2, 3, 4, 5, 6, 7, 8) or a decimal, preferably 2, 4, 6, 8, and more preferably 2.

[0139] In another aspect, the present application provides a pharmaceutical preparation (eg, a pharmaceutical composition) comprising the antibody-drug conjugate of the present application and a pharmaceutically acceptable diluent, carrier, or excipient.

[0140] In another aspect, the present application provides a medical preparation comprising the antibody-drug conjugate of the present application. In some embodiments, the medical preparation is in the form of a kit comprising a container for containing the antibody-drug conjugate of the present application. In one embodiment, the medical preparation further comprises printed instructions for using the preparation in a method for treating or preventing cancer (particularly cancer expressing ROR1).

[0141] The antibody-drug conjugates provided herein can effectively treat and / or prevent cancers associated with cells expressing ROR1. As non-limiting examples, cancer can be gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer, or gallbladder cancer, and metastases of the above cancers, particularly gastric cancer metastases, peritoneal metastases, and lymph node metastases. Cancers suitable for treatment with the antibody-drug conjugates provided herein can be adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary. The antibody-drug conjugates provided herein are particularly suitable for the treatment of gastric cancer, pancreatic cancer, breast cancer, and bladder cancer.

[0142] In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), T-cell leukemia (TCL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, multiple myeloma (MM), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), or non-Hodgkin's lymphoma (NHL) with Richter's transformation.

[0143] In some embodiments, the cancer to be treated can also be selected from, for example, lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B-cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, osteosarcoma, renal cell carcinoma, hepatocellular carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, multiple myeloma, brain cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, liver cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.

[0144] Therefore, the present application also provides multiple inventions related to the above-mentioned therapeutic uses.

[0145] In one aspect, the present application provides use of the above-described antibody-drug conjugate in the preparation of a medicament for treating or preventing cancer.

[0146] In another aspect, the present application provides a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the antibody-drug conjugate described above, or a pharmaceutical formulation or pharmaceutical composition comprising the antibody-drug conjugate, to the individual suffering from the cancer.

[0147] In another aspect, the present application provides use of the antibody-drug conjugate described above and an anti-proliferative agent in the preparation of a medicament for treating tumors (eg, the cancers described above).

[0148] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described above and an anti-proliferative agent.

[0149] In another aspect, the present application provides a method for treating a tumor in an individual, comprising administering a therapeutically effective amount of the antibody-drug conjugate described above, or a pharmaceutical formulation or composition comprising the antibody-drug conjugate, and an anti-proliferative agent to the individual having the tumor.

[0150] In some embodiments, antiproliferative agents include but are not limited to paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, iproplatin.

[0151] In some embodiments, the anti-proliferative agent may also be another antibody, antibody-drug conjugate, or fusion protein.

[0152] On the other hand, the application provides suitable TGase, such as mTGase, including but not limited to bacterial transglutaminase (BTG), such as EC index number EC 2.3.2.13 enzyme (protein-glutamine-γ-glutamyl transferase). In some embodiments, mTGase is from Streptomyces radaka (TG_SL, SEQ ID NO.15). In some embodiments, mTGase is from Streptomyces mobara (TG_SM, SEQ ID NO.16).

[0153] Example

[0154] In order to promote understanding of the present invention, the present invention will be described below with reference to certain embodiments and using specific language. However, it should be understood that these specific embodiments are not meant to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further direct or indirect application of the present invention, will generally be expected by those skilled in the art.

[0155] Example 1: Construction of humanized expression vector and plasmid preparation

[0156] The mouse antibody 17-F12-G8-A6 was humanized to obtain three humanized antibodies: hu17-H2L1, hu17-H3L1, and hu17-H4L1. The variable region sequences of the heavy chain and light chain of 17-F12-G8-A6 are shown in SEQ ID No. 17 and SEQ ID No. 18 in the sequence listing, respectively; the variable region sequences of the heavy chain and light chain of hu17-H3L1 are shown in SEQ ID No. 7 and SEQ ID No. 8 in the sequence listing, respectively; the variable region sequences of the heavy chain and light chain of hu17-H2L1 are shown in SEQ ID No. 10 and SEQ ID No. 8 in the sequence listing, respectively; the variable region sequences of the heavy chain and light chain of hu17-H4L1 are shown in SEQ ID No. 12 and SEQ ID No. 8 in the sequence listing, respectively; the constant region sequences of the heavy chain and light chain of hu17-H2L1, hu17-H3L1, and hu17-H4L1 are shown in SEQ ID No. 13 and SEQ ID No. 14 in the sequence listing, respectively.

[0157] Plasmid preparation:

[0158] The target sequence was cloned by PCR, and the PCR product was purified by gel electrophoresis. The target fragment was obtained using a gel extraction kit (Tiangen). The gel-extracted product was ligated with the plasmid vector pcDNA3.4 to generate a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli (E. coli) and plated onto ampicillin-resistant plates for colony screening. Colonies were randomly selected for sequencing. Positive colonies with correct sequencing results were expanded and the plasmid was extracted using an endotoxin-free plasmid extraction kit (Tiangen) for further sequencing verification.

[0159] Example 2 Humanized Monoclonal Antibody Expression

[0160] 1) Take 145×10 CHO cells 6 Centrifuge and remove the supernatant.

[0161] 2) Add approximately 0.5 mL of electroporation buffer to the cells, mix thoroughly, and then add an appropriate amount of plasmid (concentration 500 ng / uL).

[0162] 3) After thoroughly mixing the above cell and plasmid suspension, take 1 mL and add it to the electroporation tube. Place the electroporation tube into the electroporator for electroporation.

[0163] 4) After the electroporation is completed, the cells in the electroporated tube are divided into a shake flask containing 20 mL of culture medium prepared in advance and incubated statically for 40 minutes.

[0164] 5) After incubation, incubate the shake flask at 37°C, 270 rpm, and 8% CO2. After 24 hours, add feed / sodium butyrate / antibody and continue incubation for 3-7 days. On day 5, sample the culture and send it for ELISA to confirm the correct expression of humanized monoclonal antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1.

[0165] Example 3 Monoclonal Antibody Purification

[0166] 1) Using an AKTA purifier, equilibrate a 20 mL column with 1x PBS at a flow rate of 1 mL / min.

[0167] 2) Sample loading: flow rate 1 mL / min;

[0168] 3) Wash: 1x PBS, flow rate 1 mL / min, 20 mL;

[0169] 4) Elution: Sodium acetate buffer (pH 3.4) at 1 mL / min. Collect approximately 500 μL per tube. Collect 10 tubes in total. Read the absorbance at 280 nm using a NanoDrop instrument.

[0170] 5) Dialysis: Pipette the high-concentration protein into a dialysis bag and place it in a beaker of 1x PBS pH 7.0 for dialysis.

[0171] 6) Obtain monoclonal antibodies with an antibody purity (SEC-HPLC) > 95%.

[0172] Example 4 Affinity evaluation of humanized monoclonal antibodies

[0173] 1. Determination of humanized antibody affinity by biofilm interferometry (BLI)

[0174] Using an Octet RED96e instrument, the HIS capture sensor was immobilized with 5 μg / mL of different antibody samples. ROR1 antigen was serially diluted, starting at 100 nM and proceeding in a two-fold dilution series, with a total of seven steps. The buffer system was PBS pH 7.4, 0.02% Tween 20, and 0.1% BSA. The assay steps were: ① Baseline 60 s, ② Loading 30 s (antibody immobilization, threshold set at 0.50 nm), ③ Baseline 2 120 s, ④ Association 120 s, ⑤ Dissociation 300 s, and ⑥ Regenreation 30 s. The experimental results are shown in Table 1.

[0175] Table 1: Affinity test results of mouse and humanized antibodies using biomembrane interferometry

[0176] The results showed that after humanization, the humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 had affinities comparable to those of the mouse antibody 17-F12-G8-A6.

[0177] 2. Determination of humanized antibody affinity by enzyme-linked immunosorbent assay (ELISA)

[0178] ROR1 protein (ACROBiosystems, Catalog No. RO1-H5223) was incubated with various concentrations of humanized anti-ROR1 antibody samples and then incubated with a Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Thermo). The affinity of the samples for ROR1 protein was analyzed by measuring the signal at 450 nm at various concentrations using a microplate reader. The results are shown in Table 2.

[0179] Table 2 Affinity test results of mouse and humanized antibodies

[0180] The results showed that the anti-ROR1 humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 had good affinity for ROR1 protein.

[0181] 3. Determination of humanized antibody affinity by flow cytometry fluorescence activated cell sorting (FACS)

[0182] In this experiment, FACS was used to evaluate the affinity of ROR1 humanized antibody to the stably transfected cell line MDA-MB-231-ROR1 (Kangyuan Bochuang) expressing ROR1. ROR1 antigen-positive MDA-MB-231-ROR1 cells were digested with trypsin and collected. 1×10 5 Cells were plated into 96-well plates and serially diluted with antibodies (starting at 15 μg, 5-fold dilution, 11 concentration points). After incubation at 37°C for 2 hours, the cells were washed with PBS and then incubated with IgG-binding secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody) at 37°C for 1 hour. Absorbance was measured by flow cytometry and curves were fitted using GraphPad software. The results are shown in Table 3 and Figure 2.

[0183] Table 3 FACS affinity test results of mouse and humanized antibodies

[0184] The results showed that after humanization, the affinity of the anti-ROR1 humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 for the ROR1-expressing tumor cell MDA-MB-231-ROR1 was comparable to that of the mouse antibody 17-F12-G8-A6.

[0185] 4. Biomembrane Interferometry (BLI) assay for humanized antibody Fc function

[0186] Antibody Fc region affinity was determined using bio-layer interferometry (BLI) using an Octet RED96e instrument. HISIK capture sensors were immobilized with 5 μg / ml of FcRn, CD64, CD32a, CD32b, CD16 F176, CD16 V176, and C1q, respectively. The test samples were serially diluted, starting at 100 nM and proceeding in two-fold dilutions for a total of seven steps. The buffer system was PBS pH 7.4 with 0.02% Tween 20. The following steps were used: ① Baseline 60 s, ② Loading 800 s (antibody immobilization, threshold set at 0.50 nM), ③ Baseline 2 120 s, ④ Association 120 s, ⑤ Dissociation 300 s, and ⑥ Regeneration 300 s. Blank buffer was used for blank treatment, and the baseline was aligned with the y-axis. Savitzky-Golay fitting was performed in Octet data analysis software. The specific results are shown in Table 4.

[0187] Table 4 Affinity detection of humanized antibodies and FcγRs

[0188] Through point mutation design in the Fc region, the hu17-H4L1-AAAS antibody with L234A, L235A, P329A and P331S mutations in the Fc region, or the hu17-H4L1-AAKS antibody with L234A, L235A, G236K and P331S mutations in the Fc region, can attenuate CD64-mediated ADCP, C1q-mediated CDC, and CD16-mediated ADCC, without affecting the binding of the antibody to FcRn, thereby not affecting the half-life of the antibody.

[0189] Example 5 Evaluation of Thermal Stability of Humanized Monoclonal Antibodies

[0190] Differential Scanning Calorimetry (DSC) is widely used to characterize the thermal stability of high-level structures in biological products. It can measure and analyze the change in heat capacity of a sample during a controlled heating process in real time. By controlling the temperature change through a program, the relationship between the power difference (heat flow rate) of the sample and the temperature is measured. Thermodynamic parameters characterizing the thermal stability of the sample, such as the sample's Tonset (melting point temperature), Tm (melting temperature), and ΔH (enthalpy change), are then fitted using a thermodynamic model.

[0191] In this experiment, a Malven MicroCal PEAQ-DSC differential scanning calorimeter was used to detect the stability of the ROR1 humanized antibody. The parameter settings were: scan start temperature: 20°C, scan end temperature: 100°C, and scan rate: 90°C / h. The molecular thermal stability was analyzed by differential scanning calorimetry (DSC). The test results are shown in Table 5.

[0192] Table 5 DSC test results of mouse antibodies and humanized antibodies

[0193] The results showed that there was no significant difference in stability data (Tonset, Tm1, Tm2) between the humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 and the murine antibody 17-F12-G8-A6.

[0194] Example 6 ADC Preparation

[0195] 1. UC961-vcMMAE

[0196] The UC961 antibody was replaced with a 7.0% PBS / 5mM EDTA buffer to a concentration of 10 mg / mL. 3 molar equivalents of TCEP reducing agent were added according to the antibody's molar concentration, and the mixture was heated in a 37°C water bath for 1 hour. Then, 2.5 molar equivalents of vc MMAE (CAS No: 646502-53-6) were added and allowed to react at room temperature for 30 minutes. The reaction was stopped after 30 minutes by adding 2.5 molar equivalents of N-acetyl-L-cysteine. After stopping the reaction, free small molecules and other impurities were removed by ultrafiltration and concentration, yielding UC961-vc MMAE.

[0197] 2. Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 sample preparation

[0198] A specific volume of LND1002 (containing the drug and the linker NH2-PEG3-Val-Cit-pABC, see Figure 1), Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1 antibodies, mTGase (SEQ ID NO. 15, used in all following examples), and HO were added to EP tubes, sealed, mixed, and incubated at room temperature for no more than 4 days. mTGase catalyzes the formation of a stable amide bond (isopeptide bond) between LND1002 and the antibody glutamine amino acid. The conjugation reaction was considered complete when the heavy chain coupling efficiency exceeded 95% and was immediately purified.

[0199] Using a sterilized AKTA system, the coupled reactants were loaded onto a sterilized Protein A (MabSelect Sure) column at 30-35 g ADC / L resin with a minimum residence time of 5 minutes to ensure complete binding of the ADC product. The column was then washed with an excess of binding and wash buffer (1 × PBS pH 7.0) to remove mTGase, unreacted LND1002, and any unwanted buffer components prior to low pH elution of the desired product. The eluted fractions were collected in a collection tube pre-filled with neutralization buffer to obtain antibody-drug conjugates Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002.

[0200] Example 7 Determination of coupling sites of Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 samples

[0201] ADC samples were enzymatically digested with protease. The resulting peptides were analyzed using ultrahigh performance liquid chromatography (ULC) and high-resolution mass spectrometry (HRMS). The raw data were analyzed using UNIFI software within UPLC-MS / MS to identify the modification sites and relative intensities. 100 μg of ADC sample was added to a guanidine hydrochloride solution to a final concentration of 6 M, followed by 1 M dithiothreitol (DTT) to a final concentration of 20 mM. The mixture was mixed and incubated at 37°C for 90 min. After cooling to room temperature, 1 M iodoacetamide (final concentration 50 mM) was added, mixed, and incubated at room temperature in the dark for 45 min. 2 M urea (Tris-HCl, pH 7.5) was added to the upper layer of a 10 kDa ultrafiltration membrane, centrifuged at 13,000 rpm for 10 min, and the resulting solution was discarded and replaced twice. Trypsin was added at a ratio of 1:25 enzyme:protein (w:w), mixed, and incubated at 37°C for 4 h. After the reaction, 0.5 μL FA was added to the enzymatic hydrolysis solution to terminate the enzymatic hydrolysis reaction, and the supernatant was collected by centrifugation for analysis.

[0202] Ultra-high performance liquid chromatography (ULC) was performed using an ACQUITY UPLC H-Class PLUS (Waters). Parameters were: column temperature 50°C, flow rate 0.3 ml / min, detection wavelength 214 nm, mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile. A high-resolution mass spectrometer was used, using a Vion Q-TOF (Waters), in MSE acquisition mode, capillary voltage (kV) 3 V, MSE collision energy 20-45 eV, and ion source temperature 120°C. LC-MS / MS raw data were analyzed using UNIFI software.

[0203] The percentage of modified sites is calculated as follows: Percent Q(X) = Area Q(X) / (Area(X) + Area Q(X)) × 100%. Note: Percent Q(X) is the percentage of amino acid sites X where modifications occur. The test results are shown in Table 6.

[0204] Table 6 ADC coupling site modification rate

[0205] The results showed that the antibody-drug conjugates formed by enzymatically coupling humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 with LND1002 had a modification rate of 100% at Q295 (EU numbering) of the antibody heavy chain HC and the carboxyl terminus GGLQSGA of the antibody light chain LC.

[0206] Example 8 Determination of ADC Drug-Antibody Coupling Ratio (DAR)

[0207] 1. UC961-vc MMAE (VLS101)

[0208] The drug-antibody coupling ratio (DAR) distribution was determined by hydrophobic chromatography (HIC-HPLC) and the DAR value was calculated. Experimental instrument: High-performance liquid chromatograph (e2695, Waters) Chromatographic column: TSKgel Butyl-NPR, 4.6mm*10cm (Cat. No. 0042168 / TOSOH) Experimental method:

[0209] Mobile phases: Mobile phase A (20 mM PB, pH 7.0, 1.5 M (NH₄)₂SO₄ aqueous solution) and Mobile phase B (20 mM PB, pH 7.0, 25% isopropanol). The flow rate was controlled at 0.8 mL / min, the column temperature was 30°C, and the detection wavelength was 280 nm. The gradient program was 0-100% mobile phase B (0-20 minutes), 100% mobile phase B (20-25 minutes), and 100% mobile phase A (25-30 minutes). The sample with no attached small molecules was designated DAR0, the sample with two attached small molecules was designated DAR2, the sample with four attached small molecules was designated DAR4, the sample with six attached small molecules was designated DAR6, and the sample with eight attached small molecules was designated DAR8. Therefore, the elution order was DAR0, DAR2, DAR4, DAR6, and DAR8. The results were analyzed using area normalization, and the DAR value was approximately 3.9 (see Figure 3).

[0210] 2. Molecular weight verification of Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002

[0211] Molecular weight determination was performed using liquid chromatography-mass spectrometry (LC-MS). The following experimental procedures were used: a chromatographic column (Advance Bio SEC 200A 1.9 μm 2.1×15) was selected, mobile phase A was 100 mM ammonium formate, pH 7.0, and isocratic elution was performed for 12 minutes. Mass spectrometer (6230TOF, Agilent) parameters were set: Mass Range 2000-8000 m / z, Vcap 3500 V, Dry Gas Temp 300°C, Dry Gas Flow 10 L / min, Sheath Gas Temp. 325°C, Sheath Gas Flow 10 L / min. The experimental results are shown in Table 7.

[0212] Table 7: DAR value detection of Hu17-H2L1, Hu17-H3L1, Hu17-H4L1-4LND1002

[0213] The results showed that the humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 were enzymatically coupled to LND1002 to form antibody-drug conjugates with a DAR value of approximately 4.0.

[0214] Example 9: Comparison of Thermal Aggregation Temperatures of Antibodies and ADCs

[0215] Dynamic Light Scattering (DLS) technology, as an optical technology, can be used to measure the size of molecular particles in solutions and suspensions, and can also be used to analyze the dynamic behavior and structural changes of complex fluids. It has been widely used in the characterization of proteins, sugars, polymers, nanomaterials, etc.

[0216] The particle size distribution of the samples was determined using a WYATT Dyna Pro Plate Reader III high-throughput automated dynamic and static laser scattering analyzer. Before testing, the samples were gently mixed and uniformly distributed. 30 μL of the sample was pipetted into a 384-well plate, ensuring that no air bubbles were introduced and that no other particles were introduced. The 384-well plate was then placed in the instrument for testing. The following settings were used: ① Create an experiment; ② Maintain a constant temperature of 25°C; ③ DLS acquisition time of 5 seconds; and ④ DLS data acquisition frequency of 10. The results are shown in Table 8.

[0217] Table 8: Comparison of thermal aggregation temperatures between antibodies and ADCs

[0218] The results showed that after the anti-ROR1 antibody Hu17-H2L1 was obtained by enzymatic conjugation with four LND1002s, the Tagg (thermal aggregation temperature) decreased from 78.21°C to 76.84°C, a decrease of 1.37°C; after the anti-ROR1 antibody UC961 was obtained by chemical conjugation with four vc MMAEs, the thermal aggregation temperature decreased from 74.59°C to 54.2°C, a decrease of 20.39°C; the ADC obtained by enzymatic conjugation did not affect the interchain disulfide bonds of the antibody, while the ADC obtained by chemical conjugation destroyed the interchain disulfide bonds of the antibody, which had a greater impact on the thermal stability of the antibody. The ADC obtained by enzymatic conjugation with DAR4 had better thermal stability than the ADC obtained by chemical conjugation with DAR4.

[0219] Example 10: Endocytosis of ADC

[0220] MDA-MB-231 cells stably expressing ROR1 (MDA-MB-231-ROR1, Kangyuan Broad) were collected and resuspended in DMEM medium. The target cells were gently pipetted several times to resuspend into a single cell suspension. Trypan blue was used to identify cell viability and cell count. The cell density was adjusted to 1×10 5 cells / mL; 100 μL per well was inoculated into a 96-well cell culture dish, and the number of cells inoculated per well was 1×10 4 ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002) were added to a 96-well plate at a final concentration of 2 μg / mL and then incubated in a 37°C, 5% CO2 incubator for 27 hours. Cells were removed at 3, 6, 9, and 27 hours, suspended by flow cytometry, and fluorescently labeled goat anti-human Alexa Fluro 488 (Thermo) was added. ADCs remaining on the cell surface were detected by flow cytometry (using the same principle as in Example 3). The results are shown in Figure 4.

[0221] The results showed that all ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002) were internalized in MDA-MB-231-ROR1 cells, and the endocytosis rate of Hu17-H2L1-4LND1002 was better than that of UC961-vc MMAE.

[0222] Example 11: In vivo efficacy of ADC

[0223] 1. In vivo efficacy of H1975 human lung adenocarcinoma transplants

[0224] In this study, female NUNU mice of appropriate age were inoculated with H1975 cells to construct a human lung adenocarcinoma H1975 nude mouse xenograft tumor model. 3 At the time of the study, 28 animals with well-growing tumors were selected and divided into four groups balanced by tumor volume. A: 7 animals in the solvent control group were given 0.9% sodium chloride injection (0.9% INJ NS, solvent control group); B: 7 animals in the positive control group (UC961-vc MMAE); C: 7 animals in the Hu17-H2L1-4LND1002 experimental group; D: 7 animals in the Hu17-H3L1-4LND1002 experimental group. All groups were dosed once a week for a total of two times: 3 mg / kg on day 0 (D0) and 4 mg / kg on day 7 (D7). After dosing, mice were weighed and data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after dosing. The experiment ended on day 20 (D20). After asphyxiation with carbon dioxide, the mice were removed and weighed. The experimental results are shown in Table 9 and Figure 5.

[0225] Tumor inhibition rate % = (tumor weight of control group - tumor weight of experimental group) / tumor weight of control group * 100%

[0226] Table 9: In vivo efficacy of ADC against H1975 human lung adenocarcinoma xenografts

[0227] Note: ***: P < 0.001; **: P < 0.01; *: P < 0.05

[0228] Results showed that the tumor inhibition rates (TWI) for the UC961-vc MMAE (VLS101), Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 groups were 51.0%, 65.8%, and 53.7%, respectively. All groups significantly inhibited tumor growth compared to the solvent control group. The Hu17-H2L1-4LND1002 group exhibited a more pronounced inhibitory effect than the positive control UC961-vc MMAE group, while the Hu17-H3L1-4LND1002 group exhibited comparable inhibitory effects to the positive control group.

[0229] 2. In vivo efficacy in HCC1187 human breast cancer xenografts

[0230] In this experiment, female NUNU mice of appropriate age were inoculated with HCC1187 cells to establish a human breast cancer HCC1187 nude mouse xenograft tumor model. 3Twenty-eight animals with well-growing tumors were selected and divided into four groups balanced by tumor volume: A: a solvent control group of seven animals, which received intravenous 0.9% sodium chloride injection (0.9% INJ NS); B: a positive control group of seven animals, UC961-vc MMAE; C: a Hu17-H2L1-4LND1002 experimental group of seven animals; and D: a Hu17-H3L1-4LND1002 experimental group of seven animals. All groups received 5 mg / kg of the drug on days 0, 8, and 12 (dose on days 0, 8, and 12, for a total of three doses). After dosing, mice were weighed and data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after dosing. The experiment ended on day 14, and mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed. The results are shown in Table 10 and Figure 6.

[0231] Table 10: In vivo efficacy of ADC against HCC1187 human breast cancer xenografts

[0232] Note: ***: P < 0.001; **: P < 0.01; *: P < 0.05

[0233] Results showed that UC961-vc MMAE (VLS101), Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 exhibited tumor inhibition rates of 38.1%, 55.7%, and 47.7%, respectively. Compared to the solvent control group, all inhibited tumor growth. Compared to the positive control UC961-vc MMAE group, the inhibitory effects of Hu17-H2L1-4LND1002 and Hu17-H3L1-4LND1002 in the experimental groups were more significant.

[0234] Example 12: Safety Evaluation in Cynomolgus Monkeys

[0235] This study enrolled four age-appropriate cynomolgus macaques (Cynomolgus monkeys) and observed toxicity via intravenous administration of the positive control UC961-vc MMAE and the experimental test article Hu17-H2L1-4LND1002. The DAR for both the test article and the control was approximately 4. The dosing schedule is shown in the table below: Three intravenous injections were administered every three weeks. The control UC961-vc MMAE was administered at doses of 6 mg / kg, 6 mg / kg, and 6 mg / kg, respectively; the experimental test article Hu17-H2L1-4LND1002 was administered at doses of 6 mg / kg, 9 mg / kg, and 9 mg / kg, respectively. Continuous observations were performed post-dose, with general observations twice daily and detailed observations daily. Body weight was observed one day before dosing and weekly thereafter, and hematological and biochemical parameters were assessed. The detailed pre-toxicology testing schedule and results are shown in Table 11.

[0236] Table 11: Experimental design and results of safety evaluation in cynomolgus monkeys

[0237] The results of the safety evaluation experiment showed that the ADC Hu17-H2L1-4LND1002 of the present application was significantly less toxic than the positive control UC961-vc MMAE.

[0238] Example 13: Cynomolgus Monkey TK Study

[0239] Cynomolgus monkeys were given a single dose of 6 mg / kg of the test article Hu17-H2L1-4LND1002 (represented by H2L1-LND in the figure) or the positive control UC961-vcMMAE (represented by VLS in the figure). Blood was collected immediately (within 1 minute), 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, 336 hours, and 504 hours after administration to monitor the amount of ADC and free MMAE. The results are shown in Tables 12 and 13, Figures 7 and 8.

[0240] The concentration of small molecules in plasma was determined using LC-MS / MS. The assay method was as follows: 50 μL of the sample to be processed was added to 200 μL of the internal standard working solution under light-shielding conditions. The sample was vortexed at 2500 rpm for 3 minutes at room temperature. Centrifuged at 12000 rpm for 5 minutes at 2-8°C. 200 μL of the supernatant was injected and analyzed at room temperature. Liquid phase conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile. Column: Waters HSS T3 2.5μm, 2.1*50mm Column, flow rate: 0.5mL / min; injection volume: 5μL; autosampler temperature: 4±5℃; column temperature: 40±5℃; elution gradient setting: 0-0.5min: A 85%; 0.5-0.7min: A 85%~45%; 0.7-1.5min: A 45%~5%; 1.5-1.51min: A 5%~85%; 1.51-2min: A 85%; mass spectrometry conditions: ion source: Electrospray Ionization (ESI); ionization mode (Ion Mode): positive ion mode; detection mode (Detection Mode): multiple reaction monitoring (MRM); spray voltage (Ion Spray Voltage): 5500V; vortex ion spray temperature (Turbo Ion Spray Temp): 550℃; curtain gas (Curtain Gas): 0. Gas): 35 psi; Collision Gas: 9 psi; Nebulizer Gas (Gas1): 60 psi; Auxiliary Gas (Gas2): 60 psi; Data acquisition time: 2.0 min.

[0241] Table 12: ADC pharmacokinetic parameters

[0242] Note: Cmax: peak concentration; AUC 0-t , AUC 0-∞ : area under the plasma concentration-time curve; CL: clearance rate; MRT: 0-t : mean residence time; Vss: volume of distribution; Tmax: time to peak; t 1 / 2 :half life

[0243] Table 13: MMAE pharmacokinetic parameters

[0244] Note: Cmax: peak concentration; AUC 0-t , AUC 0-∞ : area under the plasma concentration-time curve; CL: clearance rate; MRT: 0-t : mean residence time; Vss: volume of distribution; Tmax: time to peak; t 1 / 2 :half life

[0245] At equivalent doses, H2L1-4LND1002 and UC961-vc MMAE exhibited comparable ADC exposure, with an exposure ratio of approximately 1.0. Furthermore, H2L1-4LND1002 exhibited lower free small molecule exposure than UC961-vc MMAE, with an exposure ratio of only 0.7. This suggests that H2L1-4LND1002 exhibits comparable ADC exposure to UC961-vc MMAE, with lower free small molecule exposure compared to UC961-vc MMAE. This suggests a better safety profile for H2L1-4LND1002, and no significant accumulation of free small molecules following administration.

[0246] The above description is only a preferred embodiment, which is only used as an example and does not limit the combination of features necessary for implementing the present application. The title provided is not intended to limit the various embodiments of the present application. Terms such as "comprising", "containing" and "including" are not intended to be limiting. In addition, unless otherwise specified, plural forms are included when there is no numeral modification, and "or" and "or" mean "and / or". Unless otherwise defined herein, the meaning of all technical and scientific terms used herein is the same as that generally understood by those skilled in the art. All publications and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present application, various modifications and variations of the described methods and compositions of the present application are obvious to those skilled in the art. Although the present application is described by specific preferred embodiments, it should be understood that the present application claimed for protection should not be unduly limited to these specific embodiments. In fact, various variations of the described modes for implementing the present application that are obvious to those skilled in the relevant art are intended to be included in the scope of the appended claims.

[0247] The relevant sequences involved in this application are as follows:

[0248] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0249] All publications and patent applications cited in this specification are incorporated herein by reference, as if each independent publication or patent application is specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance understanding of the present invention, and is not intended to limit the present invention to such theory, mechanism, proof or discovery in any way. Although the present invention has been shown and described in detail in the accompanying drawings and the preceding description, the present invention should be considered to be illustrative rather than restrictive.

Claims

1. An anti-ROR1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: 1) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NOs. 1, 2 and 3 respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NOs. 4, 5 and 6 respectively; or 2) the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NOs. 1, 9 and 3 respectively, and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NOs. 4, 5 and 6 respectively; or 3) The sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are SEQ ID NOs. 1, 11 and 3, respectively; the sequences of CDR1, CDR2 and CDR3 in the light chain variable region are SEQ ID NOs. 4, 5 and 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a humanized antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is an IgG type antibody, preferably an IgG1 type antibody. 4 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antigen-binding fragment is a Fab fragment, a F(ab′) 2 fragment or a single-chain Fv fragment (scFv).

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO.7, SEQ ID NO.10 and SEQ ID NO.12, and / or the amino acid sequence of the light chain variable region is SEQ ID NO.

8.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the heavy chain of the antibody comprises a heavy chain constant region of the amino acid sequence shown in SEQ ID NO.13, and / or the light chain of the antibody comprises a light chain constant region of the amino acid sequence shown in SEQ ID No.

19.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the C-terminus of the light chain of the antibody or antigen-binding fragment thereof is connected to a glutamine-containing tag peptide, and the glutamine-containing tag peptide is selected from LQSGA, GGLQSGA and GGGLQSGA.

8. A conjugate comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 coupled to one or more drug molecules, wherein the structure of the conjugate is Ab-(LU)n, Ab represents the anti-ROR1 antibody or antigen-binding fragment thereof, L represents a linker, U represents a drug molecule, and n is an integer or decimal from 1 to 8, and n is preferably 4.

9. The conjugate of claim 8, wherein the drug molecule is an anticancer drug, for example, a cytotoxic drug, an immunopotentiator or a radioisotope.

10. The conjugate of claim 9, wherein the cytotoxic drug is selected from a tubulin inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, an antimetabolite or an antitumor antibiotic.

11. The conjugate according to claim 10, wherein The tubulin inhibitor is selected from auristatin derivatives (such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (such as DM1, DM4, Ansamitocin, Mertansine or dolastatin peptide and its derivatives); The DNA topoisomerase inhibitor is selected from camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, for example, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, lutetotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-diamino)phenyl 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide; The DNA damaging agent is selected from calicheamicin, duocarmycin, and anthramycin derivative PBD (pyrrolobenzodiazepine); The antimetabolite is selected from methotrexate, 6-mercaptopurine or 5-fluorouracil; and / or The anti-tumor antibiotic is selected from polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride).

12. The conjugate according to claim 9, wherein The immunopotentiator is selected from levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic acid or polyinosinic acid.

13. The conjugate of claim 9, wherein the radioisotope is selected from 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 60 Co or 177 Lu.

14. The conjugate according to any one of claims 8 to 13, wherein the drug molecule is coupled to the antibody or antigen-binding fragment thereof via a linker, and the linker is connected to the antibody or antigen-binding fragment thereof via a thiol group or an amino group.

15. The conjugate of claim 14, wherein the linker is independently selected from mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG) m -Val-Cit, NH2-(PEG) m -Val-Cit-pAB and NH2-(PEG) m -Val-Cit-pABC, wherein m in (PEG)m is an integer from 1 to 8, preferably, m is 3.

16. The conjugate according to any one of claims 8 to 15, wherein The antibody comprises Q295 in its heavy chain constant region, and the linker is connected to the side chain of Q295 via an amide bond; preferably, the linker is connected to the side chain of the glutamine residue in the glutamine-containing tag peptide via an amide bond.

17. A pharmaceutical composition comprising: the conjugate according to any one of claims 8 to 16, a pharmaceutically acceptable carrier, and an anti-proliferative agent.

18. The pharmaceutical composition of claim 17, wherein the antiproliferative agent is selected from paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin or iproplatin.

19. Use of the conjugate according to any one of claims 8 to 16 or the pharmaceutical composition according to claim 17 or 18 in the preparation of a medicament for treating or preventing cancer.

20. A method of treating cancer in an individual, comprising administering a therapeutically effective amount of the conjugate of any one of claims 8 to 16 or the pharmaceutical composition of claim 17 or 18 to the individual suffering from the cancer.

21. The use of claim 19 or the method of claim 20, wherein the cancer is a ROR1 positive cancer.

22. The use or method of claim 21, wherein The cancer is a solid tumor, which is gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer, preferably adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung or ovary; more preferably gastric cancer or pancreatic cancer.

23. The use or method of claim 21, wherein: The cancer is a blood tumor, preferably lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, Richter's transformed non-Hodgkin's lymphoma, T-cell leukemia, Burkitt's lymphoma, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B-cell lymphoma.

24. A medical product comprising the conjugate of any one of claims 8 to 16 or the pharmaceutical composition of claim 17 or 18.

25. The medical product according to claim 24, which is in the form of a kit, wherein the kit comprises a container containing the conjugate or the pharmaceutical composition.

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