Antibodies against the ROR1 protein and their conjugates

Abstract

This application relates to an anti-ROR1 antibody, an antibody-drug conjugate containing the antibody, a pharmaceutical composition and medical product containing the antibody-drug conjugate, and a method for producing and using the antibody-drug conjugate, wherein the antibody-drug conjugate is effective in treating and / or preventing ROR1-positive tumors, including hematological malignancies and solid tumors (e.g., cancers such as breast cancer and bladder cancer).

Description

[Technical Field]

[0001] This application relates to the biopharmaceutical field, and more specifically to antibodies, conjugates, and the use of such antibodies or conjugates for the treatment or prevention of tumors. [Background technology]

[0002] ROR1, whose formal name is 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. While it is low in expression during embryonic development, it is highly expressed in various malignant tumors and tissues, including chronic lymphocytic leukemia (CLL), acute 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 drug resistance in tumor cells, and suppressing apoptosis. The human ROR1 molecule consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain 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 domain includes a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / ThrD), and a proline-rich domain (PRD).

[0003] Research has shown that the known physiological functions of ROR1 include: (1) playing an important role in mouse growth, being expressed in the face, limbs, heart, and lungs of mice, and ROR1-deficient mice are viable but exhibit respiratory problems and die within 24 hours of birth; and (2) during embryonic development, ROR1 plays an important role in regulating the development of muscle and skeleton in the embryo, including the regulation of cell division, proliferation, and migration, and is involved in the formation of organs such as nerves, skeleton, and blood vessels.

[0004] In cancer progression, ROR1 overexpression promotes tumor growth. ROR1 binds to Wnt5a and participates in Wnt signaling, as well as interacting with signaling pathways such as EGFR and Met, promoting tumor cell growth, proliferation, and migration. ROR1 activates CK1ε (casein kinase 1) and AKT / PI3K, thereby activating the pCREB pathway and inducing cell proliferation. ROR1 enhances EGFR signaling and induces epithelial-mesenchymal transition (EMT). ROR1 is highly expressed in most hematological malignancies and solid tumors; for example, over 90% of patients with chronic lymphocytic leukemia (CLL) are ROR1+. In some solid cancer cells, such as lung cancer, pancreatic cancer, and colorectal cancer, ROR1 expression is significantly elevated and closely associated with disease progression and treatment efficacy. Therefore, ROR1 can be used as a specific target marker in the development of anti-cancer drugs.

[0005] Breast cancer remains the most common cancer among women worldwide, with the global annual incidence rate rising by 0.2% to 8%. Approximately 1.4 million women are diagnosed with breast cancer each year, and around 500,000 die from the disease. Breast cancer is the leading cause of death for women aged 40-55. Since the late 1970s, breast cancer has consistently been the leading cause of cancer among women globally. According to data released by 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 1.16 per 100,000 women. For women in the United States, the lifetime risk of developing breast cancer by age 85 is approximately 12.5%, meaning that 1 in 8 to 10 women will develop breast cancer, and the lifetime risk of death from breast cancer is approximately 3.4%. However, thanks to improvements in healthcare and the continuous development of new drugs, while the global incidence rate of breast cancer has increased by 3% annually over the past decade, the survival rate for breast cancer patients has improved by 20%. Globally, China is considered a region with a low incidence of breast cancer among women, but in recent years, the incidence rate has clearly risen. In major Chinese cities, the incidence rate has increased by 37% and the mortality rate by 38.9% over the past decade, while in rural areas, the mortality rate has increased by 39.7%. Of particular concern is that China's average annual increase rate of breast cancer is 1-2 percentage points higher than that of high-incidence countries, continuing to rise at a rate of 3-4% annually. In urban areas, it has become the cancer with the fastest-rising mortality rate, and the age of onset is gradually becoming younger. Shanghai, Beijing, Tianjin, and coastal regions, in particular, are high-incidence areas for breast cancer in China, leading the way in the incidence rate of malignant tumors among women.

[0006] Endometrial cancer, also known as uterine body cancer, is one of the three most common malignancies of the female reproductive system, and most commonly occurs in women before, during, and after menopause. With increasing life expectancy and changes in lifestyle, the incidence of endometrial cancer has steadily risen over the past 20 years, and is showing a trend towards younger onset. In Western countries, endometrial cancer is the leading cause of malignancies in the female reproductive system. In China, according to the "2015 China Malignancy Epidemic Analysis" published by the National Cancer Center in 2019, there were approximately 69,000 cases and 16,000 deaths of endometrial cancer in 2015, with an incidence rate of 10.28 per 100,000 people, accounting for 3.88% of all female malignancies. As the second most common gynecological malignancy after cervical cancer, it accounts for approximately 20-30% of all gynecological malignancies. In some developed cities, endometrial cancer is already the leading cause of gynecological malignancies.

[0007] Prostate cancer is the most common tumor in men, accounting for 13% of all cancer cases. It accounts for 7% of all cancer-related deaths and is the second leading cause of cancer death in men. Over 41 years, the age-standardized incidence of prostate cancer has increased by 1993%, while the mortality rate has decreased by 10% in the last decade. It is a heterogeneous disease, with a wide range of etiologies (clinical courses) from men who survive for long periods without symptoms to those who die from the disease or develop severe malignancies with significant morbidity.

[0008] Currently, ROR1-targeting therapies include four ADCs, one humanized monoclonal antibody, five bispecific antibodies, and four CAR-T receptor thrombiols. The most advanced clinical stage is Phase III, and no commercially available products exist. Among the ADCs currently in clinical trials, VLS101 is in Phase III clinical trials. In October 2021, VelosBio published Phase I clinical data for lymphoma treatment with VLS101, showing favorable clinical results. However, because its coupling mechanism is asynchronous, there is a safety risk in administration due to the possibility of small molecule detachment via reverse Michael addition, which can cause hematological toxicity. The market needs more ROR1-targeting therapies to meet the diverse treatment needs of patients. [Overview of the Initiative]

[0009] According to a first aspect, the present invention provides a conjugate comprising an anti-ROR1 antibody conjugated to one or more drug molecules, or an antigen-binding fragment of the antibody.

[0010] According to a second aspect, the present invention provides a pharmaceutical composition comprising the conjugate described in the first aspect and a pharmaceutically acceptable carrier.

[0011] According to a third aspect, the present invention provides the use of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect in the manufacture of a pharmacopoeci for treating or preventing cancer.

[0012] According to a fourth aspect, the present invention provides a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect to the individual suffering from the cancer.

[0013] According to a fifth aspect, the present invention provides a medical product (e.g., a kit) comprising the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect.

[0014] According to a sixth aspect, the present invention provides the use of the conjugate according to the first aspect and an anti-proliferative agent in the manufacture of a tumor therapeutic agent.

[0015] According to a seventh aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the first aspect and an anti-proliferative agent. [[ENDENDEND]]

[0016] According to an eighth aspect, the present invention provides a method for treating a tumor in an individual, comprising administering to the individual suffering from the tumor a therapeutically effective amount of the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect and an anti-proliferative agent.

[0017] According to a ninth aspect, the present invention provides an anti-ROR1 antibody or an antigen-binding fragment of said antibody, a pharmaceutical composition comprising said antibody or antigen-binding fragment, the use of said antibody or antigen-binding fragment in the manufacture of a medicament, and a method for treating a tumor / cancer using said antibody or antigen-binding fragment.

[0018] In one embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof according to the present invention comprises 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 NO: 1, 2, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light-chain variable region are SEQ ID NO: 4, 5, and 6, respectively, or 2) the sequences of CDR1, CDR2, and CDR3 of the heavy-chain variable region are SEQ ID NO: 1, 9, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light-chain variable region are SEQ ID NO: 4, 5, and 6, respectively, or 3) the sequences of CDR1, CDR2, and CDR3 of the heavy-chain variable region are SEQ ID NO: 1, 11, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light-chain variable region are SEQ ID NO: 4, 5, and 6, respectively.

[0019] In the above embodiment, the antibody may be a monoclonal antibody, a bispecific antibody, or a humanized antibody. In the above embodiment, the antibody may be an IgG type antibody, and is preferably an IgG1 type antibody. In the above embodiment, the antigen-binding fragment may be a Fab fragment, an F(ab')2 fragment, or a single-stranded Fv fragment (scFv).

[0020] In the above embodiment, the amino acid sequence of the heavy chain variable region may be selected from the group consisting of 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. In the above embodiment, the heavy chain of the antibody may include the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 13, and / or the light chain of the antibody may include the light chain constant region of the amino acid sequence shown in SEQ ID NO: 19.

[0021] In the above embodiment, a glutamine-containing tag peptide is linked to the C-terminus of the light chain of the antibody or its antigen-binding fragment, and the glutamine-containing tag peptide is selected from the group consisting of LQSGA, GGLQSGA, and GGGLQSGA.

[0022] The present invention provides a conjugate comprising an anti-ROR1 antibody or its antigen-binding fragment as described in any one of the embodiments, conjugated to one or more drug molecules. The structure of the conjugate is Ab-(LU)n, where Ab represents the anti-ROR1 antibody or its antigen-binding fragment, L represents the linker, U represents the drug molecule, and n is an integer or decimal number from 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7, 8, and n is preferably 4.

[0023] In the above embodiment, the drug molecule may be an anticancer drug, for example, a cytotoxic agent, an immunostimulant, or a radioisotope. In the above embodiment, the cytotoxic agent may be selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, and antitumor antibiotics.

[0024] In the above embodiment, the tubulin inhibitor may be selected from the group consisting of auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) and maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, mertansine, dolastatin and their derivatives), The DNA topoisomerase inhibitors include camptothecin analogs, DNA topoisomerase I inhibitors, and their derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyacuminatine, topotecan, lurtothecan, berotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2 The group consisting of -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-indro[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide may be selected. The DNA damaging agent may be selected from the group consisting of calicheamicin, duocarmycin, anthramycin derivatives PBD (pyrrolobenzodiazepine), The antimetabolite may be selected from the group consisting of methotrexate, 6-mercaptopurine, and 5-fluorouracil, and / or The antitumor antibiotic may be selected from the group consisting of polypeptide antibiotics (e.g., actinomycin D or bleomycin), and anthraquinone drugs (e.g., doxorubicin or mitoxantrone hydrochloride).

[0025] In the above embodiment, the immunostimulant may be selected from the group consisting of levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic·polycytidylic acid or polyinosinic·polyridylic acid.

[0026] In the above embodiment, the radioisotope is 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 Co, and 177 Lu, and may be selected from the group consisting of. In the above embodiment, the drug molecule and the antibody or its antigen-binding fragment may be linked via a linker, and the linker and the antibody or its antigen-binding fragment may be linked via sulfhydryl or amino.

[0027] In the above embodiment, the linker is 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 -Independently selected from the group consisting of Val-Cit-pABC, where m in (PEG)m is an integer from 1 to 8, preferably m is 3. In the above embodiment, the C-terminus of the light chain of the antibody or its antigen-binding fragment may be linked to a glutamine-containing tag peptide, the glutamine-containing tag peptide being selected from the group consisting of LQSGA, GGLQSGA, and GGGLQSGA, and the linker and the side chain of the glutamine residue in the glutamine-containing tag peptide are linked via an amide bond.

[0028] In the above embodiment, the antibody may contain Q295 in its heavy chain constant region, and the linker and the side chain of Q295 are linked via an amide bond. In some embodiments, the linker and the side chain of the glutamine residue in the glutamine-containing tag peptide are linked via an amide bond.

[0029] The present invention further provides a pharmaceutical composition comprising a conjugate according to any one of the embodiments described above, a pharmaceutically acceptable carrier, and a selectable antiproliferative agent. In the above embodiment, the antiproliferative agent may be selected from the group consisting of paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, and iproplatin.

[0030] The present invention further provides the use of the conjugate or pharmaceutical composition described in any one of the embodiments described above in the manufacture of a pharmaceutical for treating or preventing cancer.

[0031] The present invention further provides a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the conjugate or pharmaceutical composition described in any one of the above embodiments to an individual suffering from the cancer.

[0032] 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, and more preferably gastric cancer or pancreatic cancer. In some embodiments, the cancer is a hematological malignancy, preferably mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or Richter-transformed lymphoma.

[0033] In the above embodiment, the cancer may be a ROR1-positive cancer. The present invention further provides a medical product comprising a conjugate or pharmaceutical composition as described in any one of the embodiments described above. The medical product exists in the form of a kit, comprising a container for containing the conjugate or pharmaceutical composition. [Brief explanation of the drawing]

[0034] [Figure 1]This is a schematic diagram showing the structure of the Hu17-H2L1 / H3L1 / H4L1-4LND1002 ADC conjugate, an exemplary embodiment of the present invention. The left side of the diagram shows the antibody structure, and the right side shows the corresponding linker-drug structure. Each molecule is a single anti-ROR1 humanized monoclonal antibody (Hu17-H2L1 / H3L1 / H4L1), with the amino acid at position Q295 (EU numbering) of each heavy chain linked to one molecule of MMAE or an MMAE derivative via a linker. Simultaneously, the C-terminus (carboxyl terminus) of each light chain is further linked to a glutamine-containing tag peptide (GGLQSGA), which is linked to one molecule of an MMAE derivative via a linker. The linkage between the antibody and the linker is a stable amide bond (isopeptide bond), and the average ratio of the drug molecule to the antibody (DAR) is 4.0. The right side of the figure shows the structure of LND1002 before conjugation, including the drug molecule (e.g., MMAE or an MMAE derivative) and the linker NH2-PEG3-Val-Cit-pABC. After conjugation, the terminal NH2 group of the linker is linked 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, forming the "-CH2-CH2-C(=O)-NH-PEG3-Val-Cit-pABC" structure. [Figure 2] This figure shows the cellular-level affinity of mouse antibodies and humanized antibodies against MDA-MB-231-ROR1. [Figure 3] This figure shows the detection spectrum of the UC961-VC MMAE DAR. [Figure 4] This figure shows the results of endocytosis experiments with various ADCs on MDA-MB-231-ROR1 cells. [Figure 5] This figure shows the inhibitory effect curves of various ADCs on human lung adenocarcinoma H1975 mouse transplant tumors. [Figure 6] This figure shows the inhibitory effect curves of various ADCs on human breast cancer HCC1187 mouse transplant tumors. [Figure 7]This figure shows the drug blood concentration-time curves of two types of ADCs after three administrations to cynomolgus monkeys. [Figure 8] This figure shows the blood concentration-time curves of free low molecular weight drugs after three administrations of two types of ADCs to cynomolgus monkeys. [Modes for carrying out the invention]

[0035] [Definition] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as those understood by those skilled in the art. For definitions and terminology in this art, those skilled in the art can refer in particular to *Current Protocols in Molecular Biology* (Ausubel). Amino acid residue abbreviations are standard three-letter and / or one-letter codes used in this art, referring to one of 20 common L-amino acids.

[0036] Regardless of the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific embodiments are described as accurately as possible. However, all numerical values ​​inherently contain a certain degree of error due to the standard deviation present in each measurement. Furthermore, all ranges disclosed herein should be understood as covering all subranges included within that range. For example, the described range "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), i.e., all subranges starting from a minimum value of 1 or greater, e.g., 1 to 6.1, and all subranges ending at a maximum value of 10 or less, e.g., 5.5 to 10. Also, any references referred to as "incorporated herein" should be understood as incorporated as a whole.

[0037] As used herein, terms such as “pharmaceutical composition,” “compound drug,” and “drug combination” are used interchangeably and refer to a combination of at least one pharmaceutical and any pharmaceutically acceptable carrier or excipient that are combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes combinations that are temporally and / or spatially separated, insofar as they can work together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, combinations of nucleic acid molecules, and / or conjugates according to the present invention) may be administered to a subject (individual) as a whole or separately to the subject. When the components contained in the pharmaceutical composition are administered separately to the subject, the components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutical carrier is water, a buffered aqueous solution, an isotonic salt solution such as PBS (phosphate buffer), glucose, mannitol, dextrorotatory glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol and triglycerides. The type of pharmaceutical carrier used depends, in particular, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the present invention may contain humectants, emulsifiers, or buffering agents as additives.

[0038] The pharmaceutical composition, vaccine, or pharmaceutical preparation according to the present invention may be administered by any suitable route, such as oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

[0039] As used herein, the terms “therapeutic effective dose” or “effective dose” refer to a dose sufficient to demonstrate the benefit of the treatment to the subject being administered. The actual amount administered, the rate of administration, and the time course of administration will depend on the condition and severity of the subject being treated.

[0040] As used herein, the term “subject” means a mammal such as a human, but may also mean other animals such as wild animals (e.g., herons, storks, cranes), domestic animals (ducks, geese, etc.), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, tarbagans, ground squirrels).

[0041] The term "antibody" broadly encompasses intact antibodies and any antigen-binding fragments ("antigen-binding portions") or single-chain forms. A "full-length antibody / intact antibody" refers to a protein containing at least two heavy chains (H) and two light chains (L) linked to each other via disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region containing three domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region containing one domain CL. The VH and VL regions may be further subdivided into multiple hypervariable regions called complementarity-determining regions (CDRs), with more conservative regions called framework regions (FRs) interspersed between them. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (such as effector cells) and to host tissues or factors, including the first component (Clq) of the classical complement system. Chimeric antibodies or humanized antibodies are also included in the antibodies according to the present invention. Full-length antibodies / intact antibodies may be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or any of the aforementioned subclasses), but the antibody does not need to belong to any particular class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant domain of the heavy chain. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are publicly known.

[0042] The complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are known to be the regions within the variable region that have the greatest influence on the affinity and specificity of an antibody. There are many common definition methods for VH or VL CDR sequences, including Kabat, IMGT, Chothia definitions, and Contact definitions. For a given antibody's variable region amino acid sequence, the CDR amino acid sequences within the VH and VL sequences can be determined based on the Kabat, IMGT, Chothia definition, or Contact definition. In this application, unless otherwise specified, all CDR amino acid sequences described herein (e.g., SEQ ID NOs: 1-6, 9, 11) are shown according to the Kabat definition rules. However, as is well known to those skilled in the art, there are multiple methods in the art for defining antibody CDRs. While the scope of protection claimed in the claims of this application is based on sequences shown according to the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0043] The term "humanized antibody" refers to an antibody that contains the CDR region of a human-derived antibody, and in which the other parts of the antibody molecule are derived from one (or more) types of human antibodies. Furthermore, in order to maintain binding affinity, some residues in the backbone (called FR) region can be modified, and the humanized antibody or fragment thereof according to the present invention can be prepared by techniques well known to those skilled in the art.

[0044] The term "semi-humanized antibody" refers to an antibody in which one antibody chain contains a mouse variable region (similar to that in chimeric antibodies) and the other antibody chain contains a humanized variable region, compared to a humanized antibody or a fully humanized antibody.

[0045] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. The chimeric antibody or fragment thereof according to the present invention can be prepared using genetic recombination technology. For example, the chimeric antibody can be prepared by cloning recombinant DNA containing a promoter, a sequence encoding the variable region of a non-human (particularly mouse) monoclonal antibody described in the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody according to the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, and the specificity of the antibody is determined by the variable region derived from mouse DNA, and its isotype is determined by the constant region derived from human DNA. For a method of preparing a chimeric antibody, see, for example, Verhoeyn et al. (BioEssays, 8:74, 1988).

[0046] The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0047] The term "bispecific antibody" refers to an antibody that has the ability to simultaneously bind to two different antigenic epitopes. The two antigenic epitopes may be located on different antigens or on the same antigen. Bispecific antibodies may have multiple structural configurations. For example, a bispecific antibody may consist of two Fc fragments and two binding sites that fuse to each of them (similar to a native antibody except that the two arms bind to different antigenic targets or epitopes), and the antigen-binding sites may be single-chain antibodies (scFv) or Fab fragments.

[0048] As used herein, the term "antigen-binding fragment" refers in particular to antibody fragments, such as Fv, scFv (sc refers to a single strand), Fab, F(ab')2 or Fab', scFv-Fc fragments, or diabodies, or any fragment whose half-life can be extended by chemical modification or introduction into liposomes. Examples of such chemical modifications include the addition of poly(alkylene) glycol, such as polyethylene glycol ("polyethylene glycolation, PEGation") (referred to as "polyethylene glycolated fragments" of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol). The antigen-binding fragment of the anti-ROR1 antibody of this application has ROR1 binding activity. For example, the antigen-binding fragment is composed of or contains a sub-sequence of the variable chain of the heavy or light chain of the derived antibody, the sub-sequence being sufficient to maintain the same binding specificity and sufficient affinity as the derived antibody, and the antigen-binding fragment contains at least five amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of the derived antibody sequence.

[0049] Examples of antigen-binding fragments include, but are not limited to, the following specific examples: (1) a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) an F(ab')2 fragment which may be a bivalent fragment having two Fab' fragments linked by a disulfide crosslink in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having VL and VH domains of one arm of an antibody; (4) a single-stranded Fv(scFv) which may be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker; and (5) a (scFv)2 which may include two VH domains linked by a peptide linker and two VL domains bound to the two VH domains via a disulfide crosslink.

[0050] The terms "Fc fragment," "Fc domain," "Fc portion," or similar terms refer to a part of the constant region of the antibody heavy chain, including the hinge region and the CH2 and CH3 fragments of the constant region.

[0051] Typically, to prepare monoclonal antibodies or their antigen-binding fragments, particularly mouse-derived monoclonal antibodies or their antigen-binding fragments, one can refer to the techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or the techniques of Kohler and Milstein for preparation from hybridoma cells (Nature, 256:495-497, 1975).

[0052] The term "homology / identity / consistency" of an amino acid or nucleic acid sequence is defined as the proportion of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and capping, to the maximum proportion of identity as required. Methods and computer programs for alignment are known to those skilled in the art.

[0053] 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.

[0054] The term "ADC" refers to an antibody-drug conjugate, which is a device in which an active small-molecule drug is chemically bonded to an antibody or its antigen-binding fragment via a linker. In this specification, the terms "ADC," "antibody-drug conjugate," and "conjugate (complex)" are used interchangeably.

[0055] As used herein, "cancer" refers to a proliferative disorder caused by or characterized by cell proliferation, in which cells have lost their sensitivity to normal proliferative control. The term "cancer" includes tumors and other proliferative disorders. Cancers of the same histological type originate from the same tissue and are classified into different subtypes based on their biological characteristics.

[0056] The term "UC961-vc-MMAE," also known as VLS-101, is an ADC (antibody-conjugate) in which the microtubule polymerization inhibitor MMAE is conjugated to the cysteine ​​residue of the antibody Cirmtuzumab (UC961) using the cleavable linker MC-Val-Cit-PAB. Its average DAR (Diagram and Arbitration Rating) is 4. The antibody sequence can be found in sequences 3-6 of CN111587124A.

[0057] In this specification, the term "NH2-PEG" 3- Val-Cit and the term "NH2-PEG" 3- "Val-Cit-pABC" has the same meaning and refers to the following structure: [ka] In the formula, * indicates the binding site to the biologically active molecule X (e.g., MMAE). When bound to an antibody, it has the following structure. [ka] In the formula, it is linked to the antibody via an amino group (-NH-).

[0058] The term "MMAE" refers to monomethyl auristatin E, which has the following structure. [ka] When connected to a linker, "MMAE" has the following structure: [ka]

[0059] The ROR1 antibody and its conjugate according to the present invention In response to the shortcomings of the prior art, the applicant has developed an anti-ROR1 humanized antibody that binds to ROR1 on ROR1-positive cells and is efficiently internalized. This antibody is extremely suitable for ADC development. Furthermore, by using an mTgase enzyme to conjugate a drug molecule (e.g., MMAE) to the anti-ROR1 antibody, an antibody-conjugate drug (ADC) with excellent uniformity and a DAR of 4 was obtained. In subsequent ADC development, it was found that using the linker according to the present invention provides good stability to the conjugate between the linker and the drug molecule. This allows the humanized antibody to be conjugated to a small molecule drug (e.g., MMAE) via the linker, and the resulting ADC drug exhibits extremely strong toxic effects against ROR1-highly expressing cancer cells, particularly pancreatic, gastric, and lung cancer cells, and possesses excellent stability. Moreover, the ADC according to the present invention employs a unique design that introduces a GGLQSGA peptide containing glutamine at the light chain terminus, achieving a high drug load of DAR=4 and improving therapeutic efficacy without significantly increasing toxicity. In in vivo experiments, intravenous administration of an antibody-drug conjugate to nude mice with ROR1-positive gastric or pancreatic xenograft tumors resulted in dose-dependent suppression of tumor growth, demonstrating a significant overall therapeutic effect. Furthermore, the ADC drug obtained in this invention exhibited a bystander effect, further enhancing the therapeutic effect. The in vivo efficacy at the same dose surpassed that of VLS101, and in preliminary toxicity studies in cynomolgus monkeys, hematological toxicity after 9 mg / kg administration was equivalent to that of VLS101 at 6 mg / kg administration, significantly expanding the therapeutic window.

[0060] In one embodiment, the present invention provides an antibody or antigen-binding fragment that can specifically bind to ROR1. Specifically, the antibody or antigen-binding fragment 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 amino acid sequence of at least one of the CDR regions is the amino acid sequence 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, and the amino acid sequence of at least one of the CDR regions is the amino acid sequence 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.

[0061] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 1, 2, and 3, and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 4, 5, and 6.

[0062] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 1, 9, and 3, and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 4, 5, and 6.

[0063] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 1, 11, and 3, and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in SEQ ID NOs: 4, 5, and 6.

[0064] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in GFSLSTSGMG (SEQ ID NO: 20), IWWDDDK (SEQ ID NO: 21), and ARPQFITTVVAFYWYFDV (SEQ ID NO: 22), and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in ENIYSN (SEQ ID NO: 23), AAT, and QHFWGTPWT (SEQ ID NO: 6), and employs the IMGT coding scheme.

[0065] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in GFSLSTSGM (SEQ ID NO: 24), WWDDD (SEQ ID NO: 25), and PQFITTVVAFYWYFDV (SEQ ID NO: 3), and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in RASENIYSNLA (SEQ ID NO: 4), AATNLAD (SEQ ID NO: 5), and QHFWGTPWT (SEQ ID NO: 6), employing Chothia's coding scheme.

[0066] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence shown in STSGMGVA (SEQ ID NO: 26), WLAHIWWDDDKR (SEQ ID NO: 27), and ARPQFITTVVAFYWYFD (SEQ ID NO: 28), and / or (ii) the light chain variable region comprises three CDR regions, each having the amino acid sequence shown in YSNLAWY (SEQ ID NO: 29), LLVYAATNLA (SEQ ID NO: 30), and QHFWGTPW (SEQ ID NO: 31), and employs the coding scheme of Contact.

[0067] In some embodiments, the antibody or antigen-binding fragment according to the present invention is isolated. 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. In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0068] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. In some embodiments, the antibody according to the present invention is a monoclonal antibody.

[0069] In some embodiments, the antibody according to the present invention is a bispecific antibody. For example, one arm of the bispecific antibody may be an antigen-binding fragment of the anti-ROR1 antibody according to the present invention (e.g., Fab or scfv), and the other arm may be an antigen-binding fragment (e.g., Fab or scfv) that targets another antigen (e.g., another antigen target usable for ADC construction) or another ROR1 epitope (different from the ROR1-binding epitope of the anti-ROR1 antibody according to the present invention).

[0070] In some embodiments, the antibody according to the present invention is a humanized antibody and includes a semi-humanized antibody. In some embodiments, the antibody or antigen-binding fragment according to the present invention has ADCC activity. In some embodiments, the antibody or antigen-binding fragment according to the present invention has CDC activity.

[0071] In some embodiments, the antibody includes a heavy chain constant region selected from the group consisting of IgG1 subtype, IgG2 subtype, and IgG4 subtype. In some embodiments, the constant heavy chain region of the antibody may be a human IgG1 subtype, a human IgG2 subtype, a human IgG4 subtype, a mouse IgG1 subtype, or a mouse IgG2a subtype. In some embodiments, the heavy chain constant region is an IgG1 subtype, i.e., the antibody is an IgG1 type antibody.

[0072] In some embodiments, the antibody includes a light chain constant region selected from a κ subtype or a λ subtype. In some embodiments, the constant region of the antibody light chain may be a human κ subtype, a human λ subtype, a mouse κ subtype, or a mouse λ subtype. In some embodiments, the antibody has a human IgG1 heavy chain constant region and contains a mutation at one or more of the following sites: L234, L235, P329, and P331, where the residue numbers follow the EU numbering system, and for example, the mutation is one or more of the following mutations: L234A, L235A, P329A, and P331S.

[0073] In some embodiments, the antibody has a human IgG1 heavy chain constant region and contains four mutations: L234A, L235A, P329A, and P331S. In some embodiments, the antibody according to the present invention is an IgG1κ antibody.

[0074] In some embodiments, the heavy chain of the antibody includes a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 13, and / or the light chain of the antibody includes a light chain constant region of the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the heavy chain of the antibody includes a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 13, and / or the light chain of the antibody includes a light chain constant region of the amino acid sequence shown in SEQ ID NO: 14.

[0075] In some embodiments, the antibody or antigen-binding fragment according to the present invention can be used to treat or prevent cancer that overexpresses ROR1. In one embodiment, an antibody capable of binding to ROR1 binds to a native epitope of ROR1 present on the surface of living cells. In one embodiment, an antibody capable of binding to ROR1 binds to the extracellular domain of ROR1. In one embodiment, an antibody capable of binding to ROR1 binds to the extracellular region of ROR1.

[0076] In some embodiments, a full-length antibody is provided in which a glutamine-containing tag peptide is further linked at the C-terminus (carboxyl terminus) of the light chain. In some embodiments, the glutamine-containing tag peptide includes LQSGA, GGLQSGA, and GGGLQSGA.

[0077] In another aspect, the present invention provides a separable polynucleotide encoding an antibody according to the present invention. In another further embodiment, the present invention provides a combination of isolated polynucleotides, the combination comprising a polynucleotide encoding the light chain of an antibody or antigen-binding fragment according to the present invention and a polynucleotide encoding the heavy chain of an antibody or antigen-binding fragment according to the present invention.

[0078] In another aspect, the present invention provides an expression vector comprising a polynucleotide or a combination of polynucleotides according to the present invention, wherein the polynucleotide is effectively ligated to a regulatory sequence that enables the expression of the polypeptide it encodes in a host cell or a cell-free expression system.

[0079] In some embodiments of the present invention, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a phage. The prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic host cell may be, for example, a fungus such as Pichia pastoris, budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), or Trichoderma; an insect cell such as Spodoptera 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 cells according to the present invention are preferably mammalian cells, and more preferably BHK cells, CHO cells, NSO cells, or COS cells.

[0080] In another embodiment, the present invention provides an antibody-drug conjugate comprising an anti-ROR1 antibody or its antigen-binding fragment conjugated to one or more drug molecules. A description of embodiments and technical features of the anti-ROR1 antibody or its antigen-binding fragment according to the present invention is provided above. 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 should understand that if ROR1 functions as a disease target of other non-cancerous / tumor cells, the drug molecule may be selected according to the target disease. Anticancer drugs include, but are not limited to, cytotoxic drugs, immunostimulants, or radioisotopes. In some embodiments, the types of cytotoxic agents include tubulin inhibitors (e.g., alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, and antitumor antibiotics.

[0081] In some embodiments, the cytotoxic drug portion may be selected from the group consisting of, for example, antitubulin agents, DNA alkylating agents, DNA crosslinking agents, DNA insertion agents, and RNA polymerase II inhibitors. In some embodiments, the cytotoxic drug portion may be monomethyl auristatin E (MMAE), azonafide, α-amanitin, or Duocarmycin (trademark). TM ), pyrrolobenzodiazepines (PBDs), the anthracycline neomycin metabolite PNU-159682 (CAS No: 202350-68-3), and their pharmaceutically acceptable salts, esters, and analogs may be selected from the group.

[0082] In some embodiments, the tubulin inhibitor may be, but is not limited to, auristatin derivatives (e.g., Monomethyl auristatin E, MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, mertansine, or dolastatin, and their derivatives).

[0083] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or DNA topoisomerase I inhibitor, and their derivatives, e.g., DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyacuminatine, topotecan, lurtothecan, berotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4( Examples include 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-indro[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0084] In some embodiments, the DNA damaging agent may include, but is not limited to, calicheamicins, duocarmycins, and anthramycin derivatives such as PBD (pyrrolobenzodiazepine).

[0085] In some embodiments, the immunostimulant may include, but is not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosine / polycytidic acid, or polyinosine / polyuridylic acid. In some embodiments, the antimetabolite may be methotrexate, 6-mercaptopurine, or 5-fluorouracil, but is not limited to these. In some embodiments, the antitumor antibiotic may be a polypeptide antibiotic (e.g., actinomycin D or bleomycin) or an anthraquinone drug (e.g., doxorubicin or mitoxantrone hydrochloride), but is not limited to these. In some embodiments, radioactive isotopes are 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 Co, or 177 Lu is one example, but it is not limited to these.

[0086] In some embodiments, an antibody capable of binding to ROR1 is covalently linked to the drug moiety via a linker. In some embodiments, the linker may be a cleavable linker. In some embodiments, the linker is cleavable under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH less than 5.5. In some embodiments, the linker is cleavable with intracellular proteases. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the linker contains a dipeptide. In some embodiments, the dipeptide is valine (Val)-citrulline (Cit). In some embodiments, the antibody is linked to the linker via the cysteinethiol of the antibody. In one embodiment, the antibody is linked to the linker via the amino acids of the antibody (particularly the amino acids of glutamine residues).

[0087] Non-restrictive examples of linkers include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, and NH2-(PEG). m-Val-Cit, NH2-(PEG) m -Val-Cit-pAB and NH2-(PEG) m -Val-Cit-pABC is an example, where m is an integer from 1 to 8.

[0088] In some embodiments, the antibody-drug conjugate according to the present invention has the following general formula Ab-(LU)n, where Ab represents the antibody targeting ROR1 of the present invention 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, where m represents the number of PEGs and is an integer from 1 to 8 (for example, 1, 2, 3, 4, 5, 6, 7, or 8), U represents a drug (for example, DM1, DM4, MMAE, MMAF, DXD, and SN38), n represents the drug antibody ratio (DAR), where the DAR value may be an average value, any number from 1 to 8 (not limited to integers, it may also be a decimal), preferably an integer from 1 to 8 (for example, 1, 2, 3, 4, 5, 6, 7, 8) or a decimal, more preferably 2, 4, 6, 8, and even more preferably 2.

[0089] In another embodiment, the present invention provides a pharmaceutical formulation (e.g., a pharmaceutical composition) comprising an antibody-drug conjugate according to the present invention and a pharmaceutically acceptable diluent, carrier, or excipient.

[0090] In another embodiment, the present invention provides a medical preparation comprising an antibody-drug conjugate according to the present invention. In some embodiments, the medical preparation exists in the form of a kit, which includes a container for housing the antibody-drug conjugate according to the present invention. In one embodiment, the medical preparation further includes printed instructions for using the preparation in a method of treating or preventing cancer (particularly cancer expressing ROR1).

[0091] The antibody-drug conjugate according to the present invention can effectively treat and / or prevent cancers associated with cells expressing ROR1. In non-limiting examples, cancers may include 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, as well as metastatic lesions of the above cancers, particularly gastric cancer metastases, peritoneal metastases, and lymph node metastases. Cancers suitable for treatment with the antibody-drug conjugate according to the present invention may also be adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovaries, and the antibody-drug conjugate according to the present invention is particularly suitable for the treatment of gastric cancer, pancreatic cancer, breast cancer, and bladder cancer.

[0092] 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 lymphoma, multiple myeloma (MM), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), or non-Hodgkin lymphoma (NHL) that has undergone Richter transformation.

[0093] In some embodiments, the cancers to be treated may be selected from the group consisting of, for example, lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B-cell lymphoma, Burkitt lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, non-Hodgkin 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.

[0094] Therefore, the present invention further provides several inventions relating to the above-mentioned therapeutic applications. According to one embodiment, the present invention provides the use of the antibody-drug conjugate described above in the manufacture of a pharmaceutical product for treating or preventing cancer. In another embodiment, the present invention 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 preparation or pharmaceutical composition containing the antibody-drug conjugate, to an individual suffering from the cancer.

[0095] In another embodiment, the present invention provides the use of the antibody-drug conjugate and antiproliferative agent described above in the manufacture of a pharmaceutical product for treating tumors (e.g., cancer as described above). In another embodiment, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate and the antiproliferative agent described above.

[0096] In another embodiment, the present invention 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 pharmaceutical composition containing the antibody-drug conjugate, and an antiproliferative agent to an individual suffering from the tumor.

[0097] In some embodiments, the antiproliferative agent may be, but is not limited to, paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, or iproplatin.

[0098] In some embodiments, the antiproliferative agent may be another antibody, an antibody-drug conjugate, or a fusion protein. In another embodiment, the present invention provides suitable TGases, such as mTGases. These include, but are not limited to, bacterial transglutaminases (BTGs), such as the enzyme with EC index number EC2.3.2.13 (protein-glutamine-γ-glutamyltransferase). In some embodiments, the mTGase is derived from Streptomyces ladaccatus (TG_SL, SEQ ID NO: 15). In some embodiments, the mTGase is derived from Streptomyces mobaraensis (TG_SM, SEQ ID NO: 16). [Examples]

[0099] To facilitate understanding of the present invention, specific embodiments are referenced below and specific terminology is used to describe the invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the invention. Any changes and further modifications to the embodiments described herein, as well as further direct or indirect applications of the invention, are ordinarily conceivable to those skilled in the art.

[0100] [Example 1] Construction of a humanized expression vector and preparation of a plasmid The mouse antibody 17-F12-G8-A6 was humanized to obtain three humanized antibodies: hu17-H2L1, hu17-H3L1, and hu17-H4L1. The sequences of the heavy and light chain variable regions of 17-F12-G8-A6 are shown in sequence numbers 17 and 18, respectively, in the sequence listing. The sequences of the heavy and light chain variable regions of hu17-H3L1 are shown in sequence numbers 7 and 8, respectively, in the sequence listing. The sequences of the heavy and light chain variable regions of hu17-H2L1 are shown in sequence numbers 10 and 8, respectively, in the sequence listing. The sequences of the heavy and light chain variable regions of hu17-H4L1 are shown in sequence numbers 12 and 8, respectively, in the sequence listing. The sequences of the constant regions of the heavy and light chains of hu17-H2L1, hu17-H3L1, and hu17-H4L1 are shown in sequence numbers 13 and 14, respectively, in the sequence listing.

[0101] Plasmid preparation: The target sequence was cloned by PCR, the PCR product was purified by gel electrophoresis, and the target fragment was obtained using a gel recovery kit (Tenkon). The gel recovery product was ligated and transformed with the plasmid vector pcDNA3.4 to obtain a recombinant plasmid. Escherichia coli (E. coli) was transformed, seeded on an ampicillin-resistant culture plate, and colonies were selected. Colonies were randomly selected and sequenced, and positive colonies with correct sequencing results were cultured on a large scale. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (Tenkon) and validated by sequencing again.

[0102] [Example 2] Expression of humanized monoclonal antibodies 1) CHO cells 145×10 6 Individual cells were collected, and the supernatant was removed by centrifugation. 2) Approximately 0.5 mL of electroporation solution was added to the cells, and after homogeneous mixing, an appropriate amount of plasmid (concentration 500 ng / μL) was added. 3) After thoroughly and uniformly mixing the cell and plasmid suspension described above, 1 mL was taken, transferred to an electroporation tube, and electroporation was performed using an electroporator. 4) After electroporation was complete, the cells in the tubes were dispensed into a pre-prepared 20 mL shaking flask containing culture medium and incubated for 40 minutes. 5) After incubation, the shaken flasks were cultured at 37°C, 270 rpm, and 8% CO2. After 24 hours, supplements / sodium butyrate / dual antibiotics were added, and the cultures were incubated for a further 3-7 days. On day 5, samples were extracted and ELISA was performed to confirm that the humanized monoclonal antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 were correctly expressed.

[0103] [Example 3] Purification of monoclonal antibodies 1) Column equilibration: Using an AKTA purification system, the chromatography column was first equilibrated with 1×PBS (20 mL) at a flow rate of 1 mL / min. 2) Sample loading: The sample was injected at a flow rate of 1 mL / min. 3) Washing for impurities: Washed with 1×PBS (20 mL) at a flow rate of 1 mL / min. 4) Elution: Elution was performed using sodium acetate buffer (pH 3.4) at a flow rate of 1 mL / min. Approximately 500 μL of the solution was collected from each tube, and after collecting a total of 10 tubes, the absorbance value at 280 nm was read using a NanoDrop device. 5) Dialysis: The high-concentration protein-containing eluate was transferred to a dialysis bag and then dialyzed in a 1x PBS (pH 7.0) beaker. 6) Monoclonal antibodies with antibody purity (SEC-HPLC) > 95% were obtained.

[0104] [Example 4] Affinity evaluation of humanized monoclonal antibodies 1. Affinity measurement of humanized antibodies using Biolayer Interference Technology (BLI) Using an Octet RED96e instrument, different antibody samples at 5 μg / mL were immobilized on an HIS capture sensor. The ROR1 antigen was serially diluted 2-fold from an initial concentration of 100 nM to create a total of seven concentration gradients. The buffer system consisted of PBS (pH 7.4), 0.02% Tween20, and 0.1% BSA. The steps were set as follows: (1) Baseline 60 seconds (baseline), (2) Loading 30 seconds (antibody immobilization, threshold set to 0.50 nm), (3) Baseline 2 120 seconds (baseline), (4) Association 120 seconds (binding), (5) Dissociation 300 seconds (dissociation), and (6) Regeneration 30 seconds (regeneration sensor). The experimental results are shown in Table 1.

[0105] [Table 1]

[0106] As a result, it was revealed that after the antibodies were humanized, the humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 exhibited affinity equivalent to that of the mouse antibody 17-F12-G8-A6.

[0107] 2. Affinity measurement of humanized antibodies by enzyme-linked immunosorbent assay (ELISA) ROR1 protein (ACROBiosystems, product number: RO1-H5223) was incubated with anti-ROR1 humanized antibody samples at different concentrations, and then incubated with an IgG-binding secondary antibody (Goat anti-Human IgG(H+L) Cross-Adsorbed Secondary Antibody, Thermo). The affinity of the samples to the ROR1 protein was analyzed by measuring the signal values ​​at 450 nm at different concentrations using a microplate reader. The experimental results are shown in Table 2.

[0108] [Table 2]

[0109] As a result, it was revealed that the anti-ROR1 humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 exhibit good affinity for the ROR1 protein.

[0110] 3. Affinity measurement of humanized antibodies by flow fluorescence-activated cell sorting (FACS). In this experiment, the affinity between the ROR1 humanized antibody and the stable transfection strain MDA-MB-231-ROR1 (Kang Yuan Bo-sang) was evaluated using FACS. ROR1 antigen-positive cells MDA-MB-231-ROR1 were recovered by trypsin digestion and loaded into a 96-well plate at a rate of 1 × 10⁶ cells per well. 5Cells were seeded, and serially diluted antibodies (starting at 15 μg, followed by a 5-fold gradient dilution to create 11 concentration points) were added in a concentration gradient. After incubation at 37°C for 2 hours, the cells were washed with PBS. Subsequently, a secondary antibody that binds to IgG (Goat anti-Human IgG(H+L) Cross-Adsorbed Secondary Antibody) was added, and the cells were incubated at 37°C for 1 hour. Absorbance values ​​were detected using flow cytometry, and the curves were fitted using GraphPad software. The test results are shown in Table 3 and Figure 2.

[0111] [Table 3]

[0112] As a result, after humanization, the humanized anti-ROR1 antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 showed affinity for ROR1-expressing tumor cells MDA-MB-231-ROR1, and affinity equivalent to that of the mouse antibody 17-F12-G8-A6.

[0113] 4. Measurement of Fc function of humanized antibodies using Biolayer Interference Technology (BLI) Bio-Layer Interferometry (BLI) was used to detect the affinity of the antibody's Fc region. Using an Octet RED96e instrument, 5 μg / mL of FcRn, CD64, CD32a, CD32b, CD16 F176, CD16 V176, and C1q were immobilized on a HISIK capture sensor. The test samples were serially diluted 2-fold from an initial concentration of 100 nM to create a total of seven concentration gradients. The buffer system was PBS (pH 7.4) + 0.02% Tween 20. The step settings were as follows: (1) Baseline 60 seconds (baseline), (2) Loading 800 seconds (antibody immobilization, threshold set to 0.50 nm), (3) Baseline 2 120 seconds (baseline), (4) Association 120 seconds (binding), (5) Dissociation 300 seconds (dissociation), and (6) Regeneration 300 seconds (regeneration sensor). Blank processing was performed using blank buffer, the baseline was aligned to the y axis, and fitting was performed using Savitzky-Golay in Octet data analysis software. The specific results are shown in Table 4.

[0114] [Table 4]

[0115] By designing point mutations in the Fc region, hu17-H4L1-AAAS antibodies with L234A, L235A, P329A, and P331S mutations in the Fc region, or hu17-H4L1-AAKS antibodies with L234A, L235A, G236K, and P331S mutations in the Fc region, both attenuated the ADCP activity mediated by CD64, the CDC activity mediated by C1q, and the ADCC activity mediated by CD16, without affecting antibody-FcRn binding, and consequently without affecting the antibody's half-life.

[0116] [Example 5] Evaluation of the thermal stability of humanized monoclonal antibodies Differential scanning calorimetry (DSC) is widely used to evaluate the thermal stability of the higher-order structure of biological products. This method allows for real-time measurement and analysis of the specific heat change of a sample during a controllable heating process. It enables programmatic control of temperature changes and determination of the relationship between the sample's power difference (heat flux) and temperature. Furthermore, by fitting a thermodynamic model, thermodynamic characterization parameters representing the thermal stability of the test specimen can be obtained, such as the sample's Tonset (melting point), Tm (melting temperature), and ΔH (enthalpy change).

[0117] In this experiment, the stability of ROR1 humanized antibodies was detected using a Malven MicroCal PEAQ-DSC differential scanning calorimeter. The parameters were set as follows: scanning start temperature: 20°C, scanning end temperature: 100°C, scanning speed: 90°C / h. The thermal stability of the molecule was analyzed using differential scanning calorimetry (DSC), and the detection results are shown in Table 5.

[0118] [Table 5]

[0119] As a result, the humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 did not show any significant difference compared to the mouse antibody 17-F12-G8-A6 in stability data (Tonset, Tm1, Tm2).

[0120] [Example 6] Preparation of ADC 1.UC961-vcMMAE The UC961 antibody was replaced with PBS 7.0 / 5 mM EDTA buffer to a concentration of 10 mg / mL. Based on the molar concentration of the antibody, 3 molar equivalents of TCEP reducing agent were added, and the mixture was heated in a 37°C water bath for 1 hour. Subsequently, 2.5 molar equivalents of vc MMAE (CAS No: 646502-53-6) were added, and the mixture was reacted at room temperature for 30 minutes. Then, 2.5 molar equivalents of N-acetyl-L-cysteine ​​were added, and the reaction was stopped after 30 minutes. After stopping the reaction, free low molecular weight compounds and other impurities were removed by ultrafiltration, concentration, and liquid replacement to obtain UC961-vc MMAE.

[0121] 2. Sample preparation for Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002. [ka]

[0122] A fixed volume of LND1002 (containing the drug and linker NH2-PEG3-Val-Cit-pABC, see Figure 1), Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1 antibodies, mTGase (sequence number 15; this enzyme was used in all of the following examples), and H2O were added to separate EP tubes, sealed, and thoroughly mixed. The reaction was then allowed to proceed at room temperature for no more than 4 days. The mTGase catalyzed the formation of a stable amide bond (isopeptide bond) between LND1002 and the glutamic acid residues of the antibodies. When the heavy chain binding rate exceeded 95%, the binding reaction was considered complete, and the mixture was immediately purified.

[0123] Using a sterile AKTA system, the reaction product after binding was loaded onto a sterile Protein A (MabSelect Sure) column with 30-35 g / L ADC resin, and the minimum residence time was set to 5 minutes to ensure complete binding of the ADC product. Subsequently, the column was washed with an excess amount of binding / washing buffer (1 × PBS pH 7.0) to remove mTGase, unreacted LND1002, and any unwanted buffer components before low-pH elution of the desired product (target product). The eluted fractions were collected in recovery tubes pre-filled with neutralizing buffer to obtain the antibody-drug conjugates Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002.

[0124] [Example 7] Measurement of binding sites of Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 samples ADC samples were enzymatically degraded using proteases, and the enzymatically degraded peptide samples were detected using ultra-high performance liquid chromatography (UPLC) and high-resolution mass spectrometry. Raw data from liquid quality detection were analyzed using UNIFI software in ULC-MS / MS to determine the modification sites and associated modification intensities of the test samples. 100 μg of ADC sample was taken, guanidine hydrochloride solution was added to a final concentration of 6 M, then 1 M dithiothreitol (DTT) was added to a final concentration of 20 mM, and the mixture was homogenized. The mixture was reacted at 37°C for 90 minutes, cooled to room temperature, and then 1 M iodoacetamide was added to a final concentration of 50 mM. The mixture was mixed and reacted at room temperature under light protection for 45 minutes. 2 M urea (Tris-HCl, pH 7.5) was added to the upper layer of a 10 kDa ultrafiltration membrane, and the mixture was centrifuged at 13,000 rpm for 10 minutes. The waste liquid was discarded, and this substitution procedure was repeated twice. Trypsin was added in an enzyme:protein ratio of 1:25 (w:w), and after homogeneous mixing, the mixture was reacted at 37°C for 4 hours. After the reaction was complete, 0.5 μL of FA was added to the enzyme digestate to stop the enzyme digestion reaction, and the supernatant was collected by centrifugation and subjected to analysis.

[0125] For ultra-high performance liquid chromatography, an ACQUITY UPLC H-Class PLUS (Waters) was used, with the following parameters: column temperature: 50°C, flow rate: 0.3 ml / min, detection wavelength: 214 nm, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution. For high-resolution mass spectrometry, a Vion Q-TOF (Waters) was employed, with the acquisition mode set to MSE, capillary voltage (kV) to 3 V, MSE collision energy to 20-45 eV, and ion source temperature to 120°C. Raw data from liquid quality detection were analyzed using UNIFI software. The formula for calculating the percentage of modified sites is as follows: Percent Q(X) = Area Q(X) / (Area(X) + Area Q(X)) × 100% (Note: Percent Q(X) represents the percentage of modified amino acid sites X). The detection results are shown in Table 6.

[0126] [Table 6]

[0127] The results showed that in antibody-drug conjugates formed by enzymatically binding humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 to LND1002, the modification rates of Q295 (EU numbering) of the antibody heavy chain HC and the carboxyl terminal GGLQSGA of the antibody light chain LC could both reach 100%.

[0128] [Example 8] Measurement of ADC drug-antibody binding ratio (DAR) 1. UC961-vc MMAE(VLS101) The distribution of drug-antibody binding ratios (DARs) was measured using hydrophobic chromatography (HIC-HPLC), and DAR values ​​were calculated. Experimental apparatus: High-performance liquid chromatography (e2695, Waters), chromatography column: TSKgel Butyl-NPR, 4.6mm x 10cm (product number 0042168 / TOSOH)

[0129] Experimental method: Mobile phase: Mobile phase A (20 mM PB, pH 7.0, 1.5 M (NH4)2SO4 aqueous solution), Mobile phase B (20 mM PB, pH 7.0, 25% isopropanol), Flow rate: 0.8 mL / min, Column temperature: 30°C, Detection wavelength: 280 nm, Gradient program: 0-20 minutes, Mobile phase B (0-100%), 20-25 minutes, Mobile phase B (100%), 25-30 minutes, Mobile phase A (100%). We define DAR0 as a molecule without any linked small molecules, DAR2 as a molecule with two linked small molecules, DAR4 as a molecule with four linked small molecules, DAR6 as a molecule with six linked small molecules, and DAR8 as a molecule with eight linked small molecules. Therefore, elution occurs in the order of DAR0, DAR2, DAR4, DAR6, and DAR8. Result analysis: When the experimental results were analyzed using area normalization, the DAR was approximately 3.9 (see Figure 3). [ka]

[0130] 2. Molecular weight verification of Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 Molecular weight was detected by liquid chromatography-mass spectrometry (LC-MS). Experimental method: A chromatography column (Column AdvanceBio SEC 200A 1.9um 2.1×15) was selected, and isocratic elution was performed for 12 minutes using mobile phase A, 100 mM ammonium formate (pH 7.0). The parameters of the mass spectrometer (6230TOF, Agilent) were set as follows: Mass Range 2000~8000m / z, Vcap 3500V, Dry Gas Temp 300℃, Dry Gas Flow 10L / min, Sheath Gas Temp 325℃, Sheath Gas Flow 10L / min. Experimental results: The experimental results are shown in Table 7.

[0131] [Table 7]

[0132] The results showed that the DAR value of the antibody-drug conjugates formed by the enzymatic binding of humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 to LND1002 was approximately 4.0.

[0133] [Example 9] Comparison of thermal aggregation temperatures of antibody and ADC Dynamic light scattering (DLS) is an optical technique used to measure the size of molecular particles in solutions and suspensions, and is also used to analyze the dynamic behavior and structural changes of complex fluids. It is widely applied to the characterization of proteins, sugars, polymers, nanomaterials, and other materials in research.

[0134] The particle size distribution of the sample was detected using a WYATT Dyna Pro Plate Reader III high-throughput automated dynamic and static laser scattering analyzer. Before detection, the sample was slowly and uniformly mixed, and 30 μL of the sample was aspirated into a 384-well plate, taking care to avoid contamination with other particles and air bubbles during this process. The 384-well plate was then placed in the instrument and detection was performed. The procedure was as follows: (1) Experiment preparation, (2) Constant temperature experiment at 25°C, (3) DLS acquisition time of 5 seconds, and (4) Number of DLS data acquisitions of 10. The results are shown in Table 8.

[0135] [Table 8]

[0136] The results showed that after obtaining an ADC (anti-ROR1 antibody) Hu17-H2L1 by enzymatic conjugation of four LND1002 molecules, the Tagg (thermal agglutination temperature) decreased from 78.21°C to 76.84°C, a decrease of 1.37°C. In contrast, after obtaining an ADC (anti-ROR1 antibody) UC961 by chemical conjugation of four vc MMAE molecules, the thermal agglutination 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, whereas the ADC obtained by chemical conjugation disrupted the interchain disulfide bonds of the antibody, significantly affecting the thermal stability of the antibody. As a result, the ADC of DAR4 conjugated by enzymatic conjugation was more thermally stable than the ADC of DAR4 conjugated by chemical conjugation.

[0137] [Example 10] Endocytotic effect of ADC MDA-MB-231 cells (MDA-MB-231-ROR1, Kang Yuan Bo-sang) that stably express ROR1 were harvested, suspended in DMEM medium, and single-cell suspension was obtained by lightly blotting target cells several times. Cell vitality (cell viability) and cell number were measured using trypan blue, and the cell density was determined to be 1 × 10⁻⁶. 5 Adjust the concentration to cells / mL, seed 100 μL / well into a 96-well cell culture dish, and place 1 × 10⁶ cells in each well.4 Cells were seeded, and ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, Hu17-H4L1-4LND1002) were added to a 96-well plate to a final concentration of 2 μg / mL. The cells were then continuously cultured for 27 hours in an incubator at 37°C and 5% CO2. Cells were removed after 3, 6, 9, and 27 hours, blown and resuspended, and fluorescently labeled goat anti-human Alexa Fluro 488 (Thermo) was added. The ADCs remaining on the cell surface were detected using a flow cytometer (the principle was the same as in Example 3), and the experimental results are shown in Figure 4.

[0138] The results showed that all ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, Hu17-H4L1-4LND1002) underwent endocytosis within MDA-MB-231-ROR1 cells, and that the endocytosis rate of Hu17-H2L1-4LND1002 was superior to that of UC961-vc MMAE.

[0139] [Example 11] In vivo efficacy of ADC 1. In vivo efficacy of human lung adenocarcinoma transplant tumors (H1975) In this study, H1975 cells were inoculated into ripe female NUNU mice to construct a nude mouse transplant tumor model of H1975 human lung adenocarcinoma. The tumor volume was approximately 100 mm². 3At this point, 28 animals with favorable tumor growth were selected and divided equally into four groups based on tumor volume. Group A (solvent control group) of 7 animals was administered 0.9% sodium chloride injection (0.9% INJ NS), Group B (positive control group) of 7 animals was administered UC961-vc MMAE, Group C (experimental group) of 7 animals was administered Hu17-H2L1-4LND1002, and Group D (experimental group) of 7 animals was administered Hu17-H3L1-4LND1002. All groups received administration once a week for a total of two doses. The first dose (day 0, D0) was 3 mg / kg, and the second dose (day 7, D7) was 4 mg / kg. After administration, the body weight of the mice was measured and the data was recorded, and the growth of the tumors was dynamically observed by measuring the diameter of the tumors at different time points after administration. The experiment was concluded on day 20 (D20). At this point, the mice were euthanized by carbon dioxide asphyxiation, and the tumors were removed and weighed. The experimental results are shown in Table 9 and Figure 5.

[0140] Tumor suppression rate (%) = (Control group tumor weight - Experimental group tumor weight) / Control group tumor weight × 100%

[0141] [Table 9]

[0142] The results showed that the tumor suppression rates (TWI) for the UC961-vc MMAE (VLS101) group, the Hu17-H2L1-4LND1002 group, and the Hu17-H3L1-4LND1002 group were 51.0%, 65.8%, and 53.7%, respectively. Compared to the solvent control group, each group significantly suppressed tumor growth. The Hu17-H2L1-4LND1002 group showed significantly better tumor suppression than the positive control group, the UC961-vc MMAE group, while the Hu17-H3L1-4LND1002 group showed comparable tumor suppression to the positive control group.

[0143] 2. In vivo efficacy of HCC1187 against human breast cancer transplant tumors In this experiment, HCC1187 cells were inoculated into ripe female NUNU mice to construct an HCC1187 human breast cancer nude mouse transplant tumor model with a tumor volume of approximately 100 mm². 3 At this point, 28 animals with favorable tumor growth were selected and divided equally into four groups based on tumor volume. Group A (solvent control group) of 7 animals received intravenous administration of 0.9% sodium chloride injection (0.9% INJ NS), Group B (positive control group) of 7 animals received UC961-vc MMAE, Group C (experimental group) of 7 animals received Hu17-H2L1-4LND1002, and Group D (experimental group) of 7 animals received Hu17-H3L1-4LND1002. All groups received a dose of 5 mg / kg on days D0, D8, and D12 (administration days: day 0, day 8, and day 12, a total of 3 administrations). After administration, the body weight of the mice was measured and the data was recorded, and the growth of the tumors was dynamically observed by measuring the diameter of the tumors at different time points after administration. The experiment was concluded on day 14 (D14). At this point, the mice were euthanized by carbon dioxide asphyxiation, and the tumors were removed and weighed. The results are shown in Table 10 and Figure 6.

[0144] [Table 10]

[0145] The results showed that the tumor suppression rates for UC961-vc MMAE (VLS101), Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 were 38.1%, 55.7%, and 47.7%, respectively. Compared to the solvent control group, all significantly suppressed tumor growth. The inhibitory effect of the experimental groups Hu17-H2L1-4LND1002 and Hu17-H3L1-4LND1002 was more pronounced compared to the positive control group UC961-vc MMAE.

[0146] [Example 12] Safety evaluation of cynomolgus macaques In this experiment, four cynomolgus monkeys of appropriate age were used, and the positive control product UC961-vcMMAE and the test product Hu17-H2L1-4LND1002 were administered intravenously, and toxic reactions were observed. The DAR of both the test product and the control product was approximately 4. The dosage design is as shown in the table below, with intravenous injection once every three weeks for three consecutive doses. The three doses of the control product UC961-vcMMAE were 6 mg / kg, 6 mg / kg, and 6 mg / kg, respectively. The three doses of the test product Hu17-H2L1-4LND1002 in the experimental group were 6 mg / kg, 9 mg / kg, and 9 mg / kg, respectively. After administration, continuous observation was performed, with general observation twice a day and detailed observation once a day. Weight changes were observed one day before administration and weekly after administration, and hematological and blood biochemical indicators were tested. The specific plan and preliminary toxicity results of the preliminary toxicity study are shown in Table 11 below.

[0147] [Table 11]

[0148] Safety evaluation experiments showed that the toxicity of ADC Hu17-H2L1-4LND1002 according to the present invention was significantly lower than that of the positive control product UC961-vc MMAE.

[0149] [Example 13] TK study in cynomolgus monkeys Cynomolgus monkeys were administered a single dose of either the test product Hu17-H2L1-4LND1002 (indicated as H2L1-LND in the figure) or the positive control product UC961-vcMMAE (indicated as VLS in the figure) at a dose of 6 mg / kg. Blood samples were collected immediately after administration (within 1 minute), and at 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, 336 hours, and 504 hours, and the amounts of ADC and free MMAE were measured. The results are shown in Tables 12 and 13, and Figures 7 and 8.

[0150] The concentration of low molecular weight compounds in plasma is measured using LC-MS / MS. <Detection Method> Under light-shielding conditions, 200 μL of internal standard working solution was added to 50 μL of the sample to be treated, and the mixture was vortexed at 2500 rpm for 3 minutes at room temperature. Centrifugation was performed at 12000 rpm for 5 minutes at 2-8°C, and 200 μL of the supernatant was collected at room temperature. The sample was then injected and analyzed. <Liquid chromatography conditions> Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: 0.1% acetonitrile formate solution, Chromatography column: Waters Xselect(R) HSS T3 2.5μm, 2.1×50mm Column Flow rate: 0.5 mL / min Sample injection volume: 5 μL, Autosampler temperature: 4±5℃ Column temperature: 40±5℃. <Setting the dissolution gradient> 0-0.5 minutes: Mobile phase A 85%, 0.5-0.7 minutes: Mobile phase A 85%-45%, 0.7-1.5 minutes: Mobile phase A 45%-5%, 1.5 to 1.51 minutes: Mobile phase A 5% to 85%, 1.51-2 minutes: Mobile phase A 85%. <Mass spectrometry conditions> Ion source: Electrospray ionization (ESI) Ionization mode: Positive ion mode, Detection mode: Multi-reaction monitoring (MRM) Ion Spray Voltage: 5,500V Turbo Ion Spray Temp: 550℃ Curtain Gas: 35 psi, Collision gas: 9 psi, Atomizing gas (Gas1): 60 psi, Auxiliary gas (Gas2): 60 psi, Data collection time: 2.0 minutes.

[0151] [Table 12]

[0152] [Table 13]

[0153] At equivalent doses, the ADC exposure levels of H2L1-4LND1002 and UC961-vcMMAE were comparable, with an exposure ratio of approximately 1.0. On the other hand, at equivalent doses, the free low molecular weight exposure level of H2L1-4LND1002 was lower than that of UC961-vcMMAE, with an exposure ratio of only 0.7 compared to UC961-vcMMAE. These results indicate that while H2L1-4LND1002 showed comparable ADC exposure levels to UC961-vcMMAE, it had a significantly lower free low molecular weight content. Since no significant accumulation of free low molecular weight was observed after administration, it suggests that the safety profile of H2L1-4LND1002 has been improved.

[0154] The above description is merely illustrative of preferred embodiments and does not limit the combination of features necessary to carry out the invention of this application. The headings provided are not intended to limit the various embodiments of this application. Terms such as “include,” “contain,” and “incorporate” are not intended to limit. Furthermore, unless otherwise specified, plural forms are included unless numerically modified. “Or” means “and / or” and “as well as / or.” Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All published documents and patents cited herein are incorporated herein by reference. Various modifications and variations of the methods and compositions described herein will be obvious to those skilled in the art without departing from the scope and spirit of this application. Although this application has been described through certain preferred embodiments, it should be understood that the claims should not be unduly limited to these particular embodiments. In fact, various modifications of the described embodiments for carrying out this application, which will be obvious to those skilled in the art, are intended to be included in the scope of the appended claims.

[0155] The arrangement according to the present invention is as follows: [Table 14] JPEG2026528756000021.jpg104164

[0156] [Table 15]

[0157] [Table 16]

[0158] [Table 17]

[0159] Any use of any examples or illustrative expressions (e.g., “For Example”) described herein is intended solely to better illustrate the invention and not to constitute a limitation on the scope of the invention, unless otherwise noted. No expression in the specification should be construed as indicating that any element not described is essential for the practice of the invention.

[0160] All publications and patent applications cited herein are incorporated herein by reference as if each individual publication or patent application were explicitly incorporated by reference specifically and individually. Furthermore, any theories, mechanisms, proofs or discoveries described herein are intended to further facilitate the understanding of the invention and are not intended in any way to limit the invention to such theories, mechanisms, proofs or discoveries. Although the invention is shown and described in detail in the drawings and the foregoing description, the invention should be considered illustrative and not limiting.

Claims

1. An anti-ROR1 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, 1) The sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are sequence numbers 1, 2, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are sequence numbers 4, 5, and 6, respectively, or 2) The sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are sequence numbers 1, 9, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are sequence numbers 4, 5, and 6, respectively, or 3) The sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are sequence numbers 1, 11, and 3, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are sequence numbers 4, 5, and 6, respectively. Anti-ROR1 antibody or its antigen-binding fragment.

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

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody is an IgG type antibody, preferably an IgG1 type antibody.

4. The aforementioned antigen-binding fragment is the Fab fragment, F(ab'). ２ A fragment, or a single-stranded Fv fragment (scFv), The antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. The amino acid sequence of the heavy chain variable region is selected from the group consisting of 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. The antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. The heavy chain of the antibody includes a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 13, and / or the light chain of the antibody includes a light chain constant region of the amino acid sequence shown in SEQ ID NO:

19. The antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. A glutamine-containing tag peptide is linked to the C-terminus of the light chain of the antibody or its antigen-binding fragment, and the glutamine-containing tag peptide is selected from the group consisting of LQSGA, GGLQSGA, and GGGLQSGA. The antibody or antigen-binding fragment according to any one of claims 1 to 6.

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

9. The conjugate according to claim 8, wherein the drug molecule is an anticancer drug, for example, a cytotoxic agent, an immunostimulant, or a radioisotope.

10. The conjugate according to claim 9, wherein the cytotoxic agent is selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, and antitumor antibiotics.

11. The tubulin inhibitor is selected from the group consisting of auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) and maytansine alkaloid derivatives (e.g., DM1, DM4, ansamitocin, mertansine, or dolastatin and its derivatives), The DNA topoisomerase inhibitors include camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyacuminatin, topotecan, lurtotecan, berotecan, exatecan, homosilatecan, and 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone Selected from the group consisting of 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-indro[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide, The DNA damaging agent is selected from the group consisting of calichemicin derivatives, duocarmycin derivatives, and anthramycin derivatives PBD (pyrrolobenzodepine). The aforementioned antimetabolites are selected from the group consisting of methotrexate, 6-mercaptopurine, and 5-fluorouracil, and / or The aforementioned antitumor antibiotic is selected from the group consisting of polypeptide antibiotics (e.g., actinomycin D or bleomycin) and anthraquinone drugs (e.g., doxorubicin or mitoxantrone hydrochloride). The conjugate according to claim 10.

12. The aforementioned immunostimulant is selected from the group consisting of levamisole, pidotimod, imiquimod, isoprinosine, polyinosine-polycytidic acid, or polyinosine-polyuridylic acid. The conjugate according to claim 9.

13. The radioactive isotope is, ２１１ At, １３１ I, １２５ I, ９０ Y, １８６ Re, １８８ Re, １５３ Sm, ２１２ Bi, ３２ P, ６０ Co, and １７７ the conjugate according to claim 9, selected from the group consisting of Lu.

14. The conjugate according to any one of claims 8 to 13, wherein the drug molecule and the antibody or its antigen-binding fragment are linked via a linker, and the linker and the antibody or its antigen-binding fragment are linked via a sulfhydryl group or an amino group.

15. The linkers include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH ２ - (PEG) ｍ -Val-Cit, NH ２ - (PEG) ｍ -Val-Cit-pAB, and NH ２ - (PEG) ｍ Independently selected from the group consisting of -Val-Cit-pABC, where m in (PEG)m is an integer from 1 to 8, preferably m is 3. The conjugate according to claim 14.

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

17. A pharmaceutical composition comprising a conjugate according to any one of claims 8 to 16, a pharmaceutically acceptable carrier, and an antiproliferative agent.

18. The pharmaceutical composition according to claim 17, wherein the antiproliferative agent is selected from the group consisting of paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, and 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 manufacture of a medicament for treating or preventing cancer.

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

21. The use according to claim 19, or the method according to claim 20, wherein the cancer is ROR1-positive cancer.

22. The cancer is a solid tumor, and the solid tumor is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, preferably an adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung, or ovary, and more preferably gastric cancer or pancreatic cancer. The use or method according to claim 21.

23. The use or method according to claim 21, wherein the cancer is a hematological malignancy, preferably selected from the group consisting of lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkin lymphoma, Richter-transformed non-Hodgkin lymphoma, T-cell leukemia, Burkitt lymphoma, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, and marginal cell B-cell lymphoma.

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

25. The medical product according to claim 24, which exists in the form of a kit including a container for containing the conjugate or the pharmaceutical composition.

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