An antibody or antigen-binding fragment thereof against ROR1

Abstract

This invention discloses an anti-ROR1 antibody or its antigen-binding fragment, relating to the fields of biotechnology and medical technology. Ten antibodies with high affinity for the ROR1 protein were screened and can specifically bind to it. This allows for the development of ROR1 detection kits, tumor diagnostic and prognostic kits, as well as antibody-containing pharmaceutical compositions, antibody-drug conjugates, and other antibody-based therapeutic methods. Experimental verification shows that the antibodies provided by this invention have a high killing rate against tumor cells. All of the above antibodies exhibit antibody concentration-dependent cytotoxicity (ADCC), with higher antibody concentrations resulting in better anti-tumor activity. Therefore, the antibodies or their antigen-binding fragments provided by this invention show promising potential for the preparation of tumor therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical technology, and more specifically, to an antibody against ROR1 or an antigen-binding fragment thereof. Background Technology

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type I transmembrane receptor tyrosine kinase. The human ROR1 molecule consists of three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes an immunoglobulin-like domain (Ig), two cysteine-rich coiled domains (FZD), and a juxtamembrane Kringle domain. The intracellular domain contains a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / Thr), and a proline-rich domain (PRD). ROR1 is primarily expressed during embryonic development, promoting embryonic growth, but it is not expressed or is expressed at low levels in most normal tissues of adults and children.

[0003] Recent studies have shown that ROR1 is highly expressed on the surface of various solid tumors (such as lung cancer, breast cancer, colon cancer, pancreatic cancer, and ovarian cancer) and hematological malignancies (such as lymphocytic leukemia and mantle cell lymphoma). ROR1 can promote tumor cell proliferation, metastasis, and drug resistance through atypical Wnt, NF-κB, or ROR1 / RhoA / Lats / TEAD signaling pathways, and is closely related to poor prognosis in malignant tumors. Therefore, ROR1 has become a promising new target in the field of cancer therapy. The development of antibodies and other drugs targeting ROR1 has significant clinical value and importance.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody against ROR1 or its antigen-binding fragment to solve the above-mentioned technical problems.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides an antibody against ROR1 or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a complementarity-determining region CDR-VH1, a complementarity-determining region CDR-VH2, and a complementarity-determining region CDR-VH3, and the light chain variable region includes a complementarity-determining region CDR-VL1, a complementarity-determining region CDR-VL2, and a complementarity-determining region CDR-VL3; the antibody or the antigen-binding fragment thereof has any one of the following sequences 1-11:

[0008]

[0009] Through screening, the inventors discovered that the aforementioned 10 antibodies or their antigen-binding fragments possess high affinity for the ROR1 protein and can specifically bind to it. This allows for the development of ROR1 detection kits, tumor diagnostic kits, and tumor prognostic kits. Experimental verification has shown that the 10 antibodies or their antigen-binding fragments provided by this invention have a high killing rate against tumor cells. All of these antibodies exhibit antibody-dependent cytotoxicity (ADCC), with higher antibody levels resulting in better anti-tumor activity. Therefore, the antibodies or their antigen-binding fragments provided by this invention show promising potential for the preparation of drugs for treating tumors.

[0010] In this invention, "ROR1" and "receptor tyrosine kinase-like orphan receptor 1" refer to the same thing.

[0011] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain backbone region and a light chain backbone region. The heavy chain backbone region includes FR-VH1, FR-VH2, FR-VH3, and FR-VH4; the light chain backbone region includes FR-VL1, FR-VL2, FR-VL3, and FR-VL4. The structure of its heavy chain variable region + heavy chain backbone region is as follows:

[0012] FR-VH1-CDR1-FR-VH2-CDR2-FR-VH3-CDR3-FR-VH4.

[0013] Its structure is: light chain variable region + light chain backbone region.

[0014] FR-VL1-CDR1-FR-VL2-CDR2-FR-VL3-CDR3-FR-VL4.

[0015] The sequences of the heavy chain backbone region and the light chain backbone region are as follows:

[0016]

[0017] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which includes a light chain constant region and a heavy chain constant region. The type of the light chain constant region is a κ-type or λ-type light chain constant region; the type of the heavy chain constant region is an IgM, IgD, IgG, IgA, or IgE heavy chain constant region.

[0018] In one alternative implementation, IgG is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, and IgG4;

[0019] In one alternative implementation, the species source of the constant region of the antibody is human, mouse, rat, cow, horse, sheep, rabbit, or dog.

[0020] The aforementioned antigen-binding fragments typically possess the same binding specificity as their source antibody and are selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv, provided they exhibit the desired antigen-binding activity. Those skilled in the art will readily understand from the description of this invention that the aforementioned antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0021] The aforementioned antibody or antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0022] In one alternative embodiment, the antibody is selected from at least one of multispecific antibodies, fusion antibodies, and chimeric antibodies. The multispecific antibody is either a bispecific antibody or a trispecific antibody.

[0023] Definitions:

[0024] Multispecific antibodies are polymers of monoclonal antibodies that recognize different epitopes. They can bind to different targets or different epitopes of the same target, and have a higher antigen recognition ability than a single monoclonal antibody.

[0025] Fusion Antibodies: The antibodies or antigen-binding fragments provided in this invention can easily combine with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering to form new fusion molecules, such as enzymes, antimicrobial peptides, or contrast agents that can extend their half-life. In the new fusion molecule, the antibody binds directionally to its target antigen, and the portion fused with the antibody can then perform the corresponding function. Clinically, doctors want drugs to remain in the body for a sufficiently long time; however, antibodies are cleared from the blood very quickly, which is detrimental to the effectiveness of the drugs they carry. Therefore, fusing antibodies with longer-lived molecules through genetic technology can increase the duration of antibody presence in the blood, i.e., extend its half-life, thereby achieving better therapeutic effects.

[0026] The "chimeric antibody" described in this invention includes, but is not limited to, antibodies formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can mitigate the immune response induced by non-human antibodies. In other embodiments, constant regions from different sources or of different types can be adaptively recombined or optimized as needed.

[0027] Secondly, the present invention also provides the application of anti-ROR1 antibodies or their antigen-binding fragments in the preparation of products for detecting ROR1, diagnosing tumors, assessing tumor prognosis, evaluating treatment efficacy, or monitoring recurrence. The products are reagents, kits, or chips; the products for diagnosing tumors or assessing tumor prognosis use ROR1 as a diagnostic or prognostic marker.

[0028] The tumor is selected from solid tumors and / or hematologic malignancies.

[0029] By detecting the level of ROR1 in the peripheral blood of a target sample, it is possible to diagnose whether the target sample has a tumor or assess the risk of tumor, and it can also provide a reference for the poor prognosis of tumors.

[0030] Furthermore, changes in ROR1 can also indicate the progression of a patient's disease. A decrease in ROR1 concentration in the patient's blood after treatment suggests treatment success, while an increase in concentration increases the risk of tumor residue or recurrence. Therefore, the anti-ROR1 antibody provided by this invention can be used for evaluating the efficacy of cancer treatment or monitoring recurrence.

[0031] In one alternative implementation, the solid tumor includes, but is not limited to, at least one of epithelial tumors and gliomas, wherein the epithelial tumor includes, but is not limited to, at least one of melanoma, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, and squamous cell carcinoma.

[0032] In one alternative implementation, the hematologic malignancy includes, but is not limited to, at least one of lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0033] Thirdly, the present invention also provides a kit for detecting ROR1, a kit for diagnosing tumors, assessing tumor prognosis, evaluating treatment efficacy, or monitoring recurrence, wherein the kit includes the aforementioned anti-ROR1 antibody or its antigen-binding fragment; the tumor is selected from solid tumors and / or hematologic malignancies; and the kit uses ROR1 as a diagnostic or prognostic marker.

[0034] In one alternative implementation, the antibody or its antigen-binding fragment is labeled with a detectable marker;

[0035] In one alternative embodiment, the kit includes a carrier carrying an antibody or an antigen-binding fragment thereof; in another alternative embodiment, the carrier is a nanoparticle, magnetic bead, agarose gel microsphere, silica microsphere, latex microsphere, or porous material.

[0036] The carrier and antibody are connected by coupling and / or physical connection. Physical connection includes, but is not limited to, electrostatic adsorption.

[0037] In other embodiments, the porous material is selected from ELISA plates or other solid supports.

[0038] For example, activated magnetic beads can be incubated with antibodies to coat the beads with antibodies. Alternatively, antibodies can be conjugated to agarose gel microspheres or silica gel microspheres to prepare corresponding immunoaffinity adsorption materials.

[0039] In one alternative embodiment, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0040] Magnetic beads, also known as magnetic microspheres, are composite microspheres with specific magnetic properties formed by combining organic polymers and inorganic magnetic nanoparticles through appropriate methods. Magnetic beads include, but are not limited to, nano-magnetic beads and micron-magnetic microspheres. In one optional embodiment, magnetic beads include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine-modified magnetic beads, silanol magnetic beads, sulfonic acid-based magnetic microspheres, thiol-based magnetic microspheres, PEG-modified magnetic beads, unmodified magnetite beads, monodisperse silica-coated magnetic beads, epoxy-based magnetic beads, monodisperse mesoporous silica-coated magnetic beads, gold-coated magnetic nanoparticles, streptavidin-modified magnetic beads, polylysine-modified magnetic beads, nickel magnetic beads, magnetic polystyrene microspheres, and silica magnetic microspheres.

[0041] Latex microspheres are spherical polymer particles in the colloidal size range formed from amorphous polymers (usually polystyrene), such as particles with a diameter of less than 100 nm, or particles with a particle size of 0.3-0.5 µm, or particles with a particle size of more than 1 µm.

[0042] Latex microspheres include, but are not limited to, latex microspheres with at least one functional group selected from hydroxyl, carboxyl, or Toysal on their surface.

[0043] In one alternative embodiment, the kit further includes a buffer solution, a positive control, and a washing solution.

[0044] In one alternative embodiment, the detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and colloids.

[0045] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0046] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0047] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0048] In one alternative embodiment, the chemiluminescent reagent is selected from at least one of acridine ester, luminol, luciferin, luteolin, ruthenium bipyridine, dioxane, rofen, isoluminol, and peroxyoxalate.

[0049] Colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0050] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0051] In one alternative embodiment, the solid tumor includes, but is not limited to, at least one of epithelial tumors and gliomas, wherein the epithelial tumor is selected from at least one of melanoma, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, and squamous cell carcinoma.

[0052] In one alternative implementation, the hematologic malignancy includes, but is not limited to, at least one of lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0053] Fourthly, the present invention also provides the use of anti-ROR1 antibodies or their antigen-binding fragments, or chimeric antigen receptors including the aforementioned anti-ROR1 antibodies or their antigen-binding fragments, in the preparation of anti-tumor drugs, wherein the tumor is selected from solid tumors and / or hematologic malignancies. The antibodies provided by the present invention have good tumor cell killing effects and can be used to prepare anti-tumor drugs.

[0054] In one alternative embodiment, the solid tumor is selected from at least one of epithelial tumors and gliomas, wherein the epithelial tumor is selected from at least one of melanoma, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, and squamous cell carcinoma.

[0055] Gastrointestinal cancers are selected from esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, bile duct cancer, small bowel cancer, colorectal cancer, and anal cancer. Colorectal tumors include one or more of the following types: polypoid, stricture, and ulcerative.

[0056] In one alternative implementation, adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0057] In one alternative implementation, the adenoma includes, but is not limited to, cystic adenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma.

[0058] In one alternative embodiment, breast cancer includes, but is not limited to, ductal carcinoma, epithelial carcinoma, or lobular carcinoma. Breast cancer, preferably stage II to IV and / or poorly differentiated aggressive ductal carcinoma, comedo carcinoma, and medullary carcinoma (preferably grade 2).

[0059] Ovarian cancer, serous and mucinous carcinoma (preferably stage Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0060] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0061] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0062] Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.

[0063] Squamous cell carcinoma includes one or more of the following: oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, esophageal squamous cell carcinoma (e.g., esophageal squamous cell carcinoma), squamous cell carcinoma of the lip, uterine squamous cell carcinoma, vaginal squamous cell carcinoma, and skin squamous cell carcinoma.

[0064] Oral squamous cell carcinoma (OSCC) is also known as oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include, but are not limited to, tongue squamous cell carcinoma.

[0065] In other embodiments, the aforementioned squamous cell carcinoma can also be squamous cell carcinoma formed by squamous metaplasia of the bronchus, bladder, renal pelvis, etc.

[0066] In one alternative implementation, the hematologic malignancy includes, but is not limited to, at least one of lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0067] Lymphocytic leukemia, for example, is selected from chronic lymphocytic leukemia or acute lymphoblastic leukemia.

[0068] Fifthly, the present invention also provides a nucleic acid molecule that encodes the aforementioned anti-ROR1 antibody or its antigen-binding fragment.

[0069] Considering the degeneracy of codons, the gene sequence encoding the above antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0070] In addition, the present invention also provides a recombinant vector comprising the above-described nucleic acid molecules.

[0071] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged into a virus) into the host cell through transformation, transfection, or transduction to express the target gene.

[0072] One type of vector is the plasmid, a circular double-stranded DNA molecule, which ligates the target DNA fragment into the plasmid circle. Another type of vector is the viral vector, which ligates and packages the target DNA fragment into the viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, and oncolytic virus). Once these vectors enter the host cell, they can express the target gene.

[0073] In a sixth aspect, the present invention also provides a cell comprising secreting the aforementioned anti-ROR1 antibody or its antigen-binding fragment and / or containing the aforementioned nucleic acid molecule.

[0074] The present invention also provides a cell comprising the above-described recombinant vector.

[0075] The host cell is selected from at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages;

[0076] In one alternative implementation, the prokaryotic host cell is Escherichia coli, Streptomyces, Bacillus subtilis, or Mycobacterium.

[0077] In one alternative implementation, the eukaryotic host cell is an animal cell, a plant cell, or a fungus;

[0078] In one alternative embodiment, the animal cells are selected from mammalian cells, insect cells, or Caenorhabditis elegans.

[0079] The mammalian cells are selected from any one of the following: 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells. Among them, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those skilled in the art.

[0080] In a seventh aspect, the present invention also provides a composition or antibody. Drug conjugates, comprising the aforementioned anti-ROR1 antibody or its antigen-binding fragment.

[0081] In a preferred embodiment of the present invention, the composition is a pharmaceutical composition;

[0082] In a preferred embodiment of the present invention, the composition further includes pharmaceutically acceptable excipients.

[0083] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in this invention, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, etc.) to prepare the antibody or its functional fragment, for example, by isolating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0084] The present invention also provides a composition comprising an antibody against ROR1 or an antigen-binding fragment thereof and pharmaceutically acceptable excipients.

[0085] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents, and flavorings.

[0086] Pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers, excipients, or solvents. Pharmaceutically acceptable excipients include a variety of organic or inorganic carriers and / or excipients because they are commonly used for pharmaceutical purposes, particularly for solid drug formulations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, talc, and sodium lauryl sulfate; and flavorings such as citric acid, menthol, and glycine. Orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0087] In a preferred embodiment of the present invention, the dosage form of the drug is tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection or spray.

[0088] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0089] In a preferred embodiment of the invention, the drug is formulated for oral or injectable administration. Injectable administration includes, but is not limited to, subcutaneous injection, intramuscular injection, intravenous injection, and intradermal injection.

[0090] The present invention has the following beneficial effects:

[0091] This invention screened and obtained 10 antibodies that have high affinity for the ROR1 protein and can specifically bind to it. This allows for the development of ROR1 detection kits, tumor diagnostic kits, tumor prognostic kits, and kits for efficacy evaluation or recurrence monitoring. Experimental verification showed that the antibodies provided by this invention have a high killing rate against tumor cells. All of the above antibodies can produce antibody concentration-dependent cytotoxicity (ADCC), with higher antibody concentrations resulting in better anti-tumor activity. Therefore, the antibodies or their antigen-binding fragments provided by this invention show promising potential for the preparation of drugs for treating tumors.

[0092] The ROR1 detection kit and tumor diagnostic kit provided by this invention have the advantages of good detection or diagnosis specificity, high sensitivity, and good repeatability. Attached Figure Description

[0093] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0094] Figure 1 A statistical chart showing the results of antibody anti-solid tumor activity assays for numbers 1-10;

[0095] Figure 2 A statistical chart showing the results of antibody anti-hematologic tumor activity assays numbered 1-10;

[0096] Figure 3 This is a statistical chart showing the results of ELISA experiments on the specific binding of 10 ROR1 antibodies to the ROR1 antigen. Detailed Implementation

[0097] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0098] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0099] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0100] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0101] Example 1

[0102] This embodiment provides a method for preparing antibodies and sequences the antibody sequences.

[0103] 1) Antibody preparation

[0104] The extracellular region sequence of the ROR1 protein was cloned into the pCDH-CMV lentiviral vector. The correctly sequenced plasmid and lentiviral packaging plasmid were transiently transfected into 293-T cells. After 48 hours, the viral supernatant was collected and used to infect lung cancer H1299 cells. The expression of ROR1 was detected by flow cytometry, which proved that ROR1 could be stably overexpressed in the cell line. Then, 8-week-old Balb / c mice were immunized by intraperitoneal injection of the ROR1-overexpressing cell line. Re-immunizations were performed on days 21 and 35 after the first immunization, with the same cell pretreatment and injection site as the first immunization.

[0105] Myeloma cells and spleen cells (prepared from the spleen of mice after booster immunization) were fused using PEG and then cultured in HAT selection medium. When the cell clones grew to one-third of the culture wells, the supernatant was aspirated, and positive hybridoma cell lines secreting ROR1 monoclonal antibodies were screened using ELISA. Then, the positive clones were subcloned using limiting dilution (2-3 times), followed by further screening by flow cytometry for hybridoma cells that stably secrete antibodies binding to the natural conformation of ROR1.

[0106] After purifying the antibodies in the hybridoma cell supernatant, 10 anti-ROR1 antibodies were obtained.

[0107] 2) Obtain antibody sequence

[0108] RNA was extracted from hybridoma cells, and the nucleic acid sequences encoding antibodies VH and VL were amplified using 5'RACE technology. The sequences were then cloned into a T vector and sequenced to obtain the sequence of each antibody.

[0109] The VL sequence of the ROR1 antibody is as follows:

[0110]

[0111] The VH sequence of the ROR1 antibody is as follows:

[0112]

[0113] Example 2

[0114] This embodiment tests the affinity of the 10 antibodies from Example 1.

[0115] The extracellular domain (Frizzled domain) of ROR1 was expressed and purified in vitro using a eukaryotic expression system. The binding of the antibody to the ROR1 extracellular domain was detected by SPR. A carboxymethylated dextran sensor chip was inserted into a surface plasmon resonance (SPR) instrument, and the system was equilibrated to optimize and determine the optimal buffer solution conditions and concentrations. A new sensor chip was then inserted, and a 1:1 mixture of N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) solution was injected at a flow rate of 10 μL / min for 10 minutes. The ROR1 antigen was diluted to 20 μg / mL with sodium acetate buffer (pH 5.0, 10 mM) and immobilized on dextran carboxylate. All unactivated dextran carboxylate was blocked with ethanolamine solution (1 M). The antibody was then injected under optimal buffer conditions. After system equilibration, data were collected and analyzed. The above antibody and human ROR1 extracellular domain showed a dissociation constant KD ≤ 10 × 10⁻⁶. -10 The affinity binding of M. Specific affinity data are as follows:

[0116]

[0117] Example 3

[0118] This embodiment tests the anti-solid tumor activity of the 10 antibodies provided in Example 1.

[0119] The killing ability of antibodies against SK-Hep1 hepatocellular carcinoma cells was evaluated using antibody-dependent cell-mediated cytotoxicity (ADCC). Peripheral blood cells (PBMCs) were isolated from human peripheral blood and mixed with SK-Hep1 hepatocellular carcinoma cells (target cell:effect cell ratio = 1:5). After incubation with the aforementioned ROR1 antibody, the cells were analyzed using Promega's CytoTox 96 commercial kit. ® The Non-Radioactive Cytotoxicity Assay detects lactate dehydrogenase (LDH) released after tumor cell death, thereby calculating the tumor cell killing rate (100%).

[0120] Results reference Figure 1 As shown, the ROR1 antibodies provided by this invention all exhibit antibody concentration-dependent cytotoxicity. Therefore, the antibodies provided by this invention possess good solid tumor-killing activity.

[0121] Example 4

[0122] Similarly, the anti-hemorrhagic activity of the 10 antibodies provided in Example 1 was tested. PBMCs were isolated from donated healthy human peripheral blood, mixed with B-cell acute leukemia cells (BALL-1) (effective cell:target cell ratio = 5:1), and then incubated with 10 ng / ml of the 10 antibodies from Example 1. The mixture was then analyzed using CytoTox 96. ® The Non-Radioactive Cytotoxicity Assay detects lactate dehydrogenase (LDH) released after tumor cell death, thereby calculating the tumor cell killing rate (100%).

[0123] Results reference Figure 2 As shown, the ROR1 antibodies provided by this invention can all produce ADCC effects. Therefore, the antibodies provided by this invention have good hematologic tumor-killing activity.

[0124] Example 5

[0125] This embodiment uses ELISA to determine the specific binding of 10 antibodies provided in Example 1 to the antigen ROR1. Specifically, the extracellular domain of ROR1, recombinantly expressed in vitro, was used as the antigen and coated onto 96-well plates, which were then blocked with 5% skim milk. The plates were then incubated with different concentrations of the 10 ROR1 antibodies from Example 1, followed by incubation with the corresponding secondary antibodies. Finally, TMB chromogenic buffer was added for color development, and the absorbance at 450 nm was measured.

[0126] Results reference Figure 3 As shown, the ROR1 antibodies provided by this invention can all specifically bind to the ROR1 antigen. Therefore, the antibodies provided by this invention have good ROR1 recognition ability.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against receptor tyrosine kinase-like orphan receptor 1 (ROR1), characterized in that, The antibody or its antigen-binding fragment has a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes complementarity-determining regions CDR-VH1, CDR-VH2, and CDR-VH3, and the light chain variable region includes complementarity-determining regions CDR-VL1, CDR-VL2, and CDR-VL3; the antibody number is selected from antibody number 5 or antibody number 7, and the CDR sequence of antibody number 5 or antibody number 7 is shown below: 。 2. The anti-ROR1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain backbone region and a light chain backbone region, wherein the heavy chain backbone region includes FR-VH1, FR-VH2, FR-VH3 and FR-VH4; and the light chain backbone region includes FR-VL1, FR-VL2, FR-VL3 and FR-VL4. The sequences of the heavy chain backbone region and the light chain backbone region are as follows: 。 3. The anti-ROR1 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment further includes a constant region, which includes a light chain constant region and a heavy chain constant region. The type of the light chain constant region is a κ-type or λ-type light chain constant region. The type of the heavy chain constant region is an IgM, IgD, IgG, IgA, or IgE heavy chain constant region. The IgG is selected from any one of IgG1, IgG2, IgG3, and IgG4.

4. The anti-ROR1 antibody or its antigen-binding fragment according to claim 3, characterized in that, The species source of the constant region of the antibody is human, mouse, rat, cow, horse, sheep, rabbit or dog.

5. The anti-ROR1 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antigen-binding fragment is selected from any one of the antibody's Fab', Fab, F(ab')2, scFv, and Fv.

6. The anti-ROR1 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody is selected from chimeric antibodies.

7. The use of the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1-6 in the preparation of products for detecting ROR1, diagnosing tumors, assessing tumor prognosis, evaluating tumor treatment efficacy, or monitoring tumor recurrence, characterized in that... The product is a reagent, kit, or chip; the product for diagnosing tumors or tumor prognosis uses ROR1 as a diagnostic or prognostic marker. The tumor is selected from lung cancer, breast cancer, liver cancer, colorectal cancer, or lymphocytic leukemia.

8. A kit for detecting ROR1, diagnosing tumors, assessing tumor prognosis, evaluating tumor treatment efficacy, or monitoring tumor recurrence, characterized in that, The kit includes an antibody against ROR1 as described in any one of claims 1-6 or an antigen-binding fragment thereof; the tumor is selected from lung cancer, breast cancer, liver cancer, colorectal cancer or lymphocytic leukemia; the kit uses ROR1 as a diagnostic marker or prognostic marker.

9. The reagent kit according to claim 8, characterized in that, The antibody or its antigen-binding fragment is labeled with a detectable marker.

10. The reagent kit according to claim 8, characterized in that, The kit includes a carrier carrying the antibody or its antigen-binding fragment. The carrier is a nanoparticle, magnetic bead, agarose gel microsphere, silica microsphere, latex microsphere, or porous material. The porous material is selected from ELISA plates.

11. The reagent kit according to claim 9, characterized in that, The detectable marker is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and colloids.

12. The use of the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1-6, or a chimeric antigen receptor including the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1-6, in the preparation of a tumor-treating drug, characterized in that, The tumor is selected from lung cancer, breast cancer, liver cancer, colorectal cancer, or lymphocytic leukemia.

13. A nucleic acid molecule, characterized in that, It encodes the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1-6.

14. A cell characterized in that, It secretes an antibody against ROR1 as described in any one of claims 1-6 or an antigen-binding fragment thereof and / or contains a nucleic acid molecule as described in claim 13.

15. A composition, characterized in that, It includes the anti-ROR1 antibody or its antigen-binding fragment as described in any one of claims 1-6.

16. The composition according to claim 15, characterized in that, The composition is a pharmaceutical composition.

17. The composition according to claim 16, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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