An antibody or antigen-binding fragment thereof against CK17, antibody conjugate, nucleic acid, vector, cell, and use thereof

Abstract

The application discloses an antibody or antigen binding fragment thereof against CK17, an antibody conjugate, a nucleic acid, a vector, a cell and application thereof, and relates to the technical field of antibody preparation.The antibody or antigen binding fragment thereof against CK17 provided by the application has good binding activity and affinity to CK17 protein, so that a detection product based on the CK17 protein marker can be developed, such as a detection reagent, a kit or a chip. The sensitivity and specificity of detection can be improved by using the antibody or antigen binding fragment thereof against CK17 to detect CK17. The antibody or antigen binding fragment thereof against CK17 can be used for early warning, diagnosis, efficacy evaluation and prognosis of diseases taking the CK17 protein as a marker. The application provides more protein selection for detection of the CK17 protein, early warning, diagnosis, efficacy evaluation and prognosis of epithelial tumors and skin diseases.

Description

An antibody against CK17 or its antigen-binding fragment, antibody-drug conjugate, nucleic acid, vector, cell, and its application. Technical Field

[0001] This invention relates to the field of antibody preparation technology, and more specifically, to an anti-CK17 antibody or its antigen-binding fragment, antibody conjugate, nucleic acid, vector, cell, and their applications. Background Technology

[0002] Cytokeratin 17 (CK17) is a type I keratin with a relative molecular weight of 46,000, playing a crucial role in the formation and maintenance of various skin appendages. CK17 is mainly distributed in the basal cells of sweat glands and the deep outer root sheath of hair follicles, and is expressed mildly in normal basal cells. Abnormal expression of CK17 can occur when epidermal keratinocytes undergo abnormal proliferation and differentiation. Multiple studies have shown that CK17 is abnormally expressed in various skin diseases and cancers, including psoriasis, seborrheic keratosis, squamous cell carcinoma of the skin, basal cell carcinoma, malignant melanoma, and other epithelial tumors. Therefore, CK17 is of significant importance for the early diagnosis, treatment monitoring, and prognosis of these diseases.

[0003] In clinical practice, immunohistochemistry (IHC) is commonly used to detect protein expression in tumor cells. The accuracy and sensitivity of IHC detection depend heavily on the quality of the monoclonal antibody that specifically binds to the protein. Therefore, developing a highly specific monoclonal antibody against the CK17 protein is of great significance for detecting CK17 protein expression levels.

[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 CK17 or its antigen-binding fragment, antibody conjugate, nucleic acid, vector, cell, and its application 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 CK17 or an antigen-binding fragment thereof, wherein the antibody includes the following complementarity-determining region:

[0008] HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1;

[0009] HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2;

[0010] HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3;

[0011] LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4;

[0012] LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5;

[0013] And LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:6.

[0014] Secondly, the present invention also provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0015] Thirdly, the present invention also provides the use of the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate in the preparation of any of the following products, wherein the product is selected from:

[0016] Reagents, kits, and chips.

[0017] Fourthly, the present invention also provides a reagent or kit for risk warning, diagnosis, efficacy monitoring, prognostic assessment, CK17 protein detection, CK17 protein enrichment, or CK17 protein purification of tumors or non-tumor skin diseases, which contains the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0018] Fifthly, the present invention also provides a nucleic acid that encodes the aforementioned antibody or its antigen-binding fragment.

[0019] In a sixth aspect, the present invention also provides a carrier comprising the aforementioned nucleic acid.

[0020] In a seventh aspect, the present invention also provides a cell comprising the aforementioned carrier.

[0021] The present invention has the following beneficial effects:

[0022] The anti-CK17 antibody or its antigen-binding fragment provided by this invention exhibits good binding activity and affinity to the target antigen (CK17 protein), thereby enabling the development of detection products based on CK17 protein biomarkers, such as detection reagents, kits, or chips. Using the anti-CK17 antibody or its antigen-binding fragment of this invention to detect CK17 can improve the sensitivity and specificity of the detection.

[0023] Antibodies against CK17 or their antigen-binding fragments can be used for early warning, diagnosis, efficacy evaluation, and prognosis of diseases using CK17 protein as a biomarker, such as epithelial tumors and skin diseases, which is beneficial for disease early warning, screening, intervention, and efficacy evaluation. Therefore, this invention provides more protein options for the detection of CK17 protein and for early warning, diagnosis, efficacy evaluation, and prognosis of epithelial tumors and skin diseases. Attached Figure Description

[0024] 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.

[0025] Figure 1 shows the results of immunohistochemical experiments on lung squamous cell carcinoma tissue. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Definition of noun

[0029] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0030] The amino acid sequence of recombinant human CK17 protein is shown in SEQ ID NO:17:

[0031] MSVRYSSSKHYSSSRSGGGGGGGGCGGGGGVSSLRISSSKGSLGGGFSSGGFSGGSFSRGSSGGGCFGGSSGGYGGLGGFGGGSFRGSYGSSSFGGSYGGIFGGGSFGGGSFGGGSFGGGGFGGGGFGGGFGGGFGGDGGLLSGNEKV TMQNLNDRLASYLDKVRALEESNYELEGKIKEWYEKHGNSHQGEPRDYSKYYKTIDDLKNQILNLTTDNANILLQIDNARLAADDFRLKYENEVALRQSVEADINGLRRVLDELLTLTKADLEMQIESLTEELAYLKKNHEEEMKDLRN VSTGDVNVEMNAAPGVDLTQLLNNMRSQYEQLAEQNRKDAEAWFNEKSKELTTEIDNNIEQISSYKSEITELRRNVQALEIELQSQLALKQSLEASLAETEGRYCVQLSQIQAQISALEEQLQQIRAETECQNTEYQQLLDIKIRLEN EIQTYRSLLEGGSSGGGGRGGGSFGGGYGGGSSGGGSSGGGHGGGHGGSSSGGGYGGGSSSGGGSSGGGYGGGSSSGGHGGSSSGGYGGGSSSGGGGGGYGGGSSGGGSSSGGGYGGGSSSGGHKSSSSSGSVGESSSKGPRYLEHHHHHH

[0032] The term “antibody” as used in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to the CK17 antigen or fragments thereof.

[0033] Typically, the variable region VH of the antibody heavy chain can be obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0034] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0035] In a first aspect, the present invention provides an antibody against CK17 or an antigen-binding fragment thereof, wherein the antibody includes the following complementarity-determining region:

[0036] HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1;

[0037] HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2;

[0038] HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3;

[0039] LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4;

[0040] LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5;

[0041] And LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:6.

[0042] HCDR1: NYWMT;

[0043] HCDR2: EIRLKSNNYGTHYAESVKG;

[0044] HCDR3:NFDY;

[0045] LCDR1: QASKSVSTSTH;

[0046] LCDR2: AIMSIS;

[0047] LCDR3: KQSQWMET.

[0048] The amino acid sequence of the complementarity-determining region described above is a novel sequence discovered and disclosed for the first time in this invention, which can endow the antibody or its antigen-binding fragment with the ability to specifically bind to the CK17 antigen. Based on the good binding activity and affinity of this binding protein, detection products based on CK17 protein biomarkers, such as detection reagents, kits, or chips, can be developed. Using the anti-CK17 antibody or its antigen-binding fragment of this invention to detect CK17 can improve the sensitivity and specificity of detection.

[0049] Antibodies against CK17 or their antigen-binding fragments can be used for early warning, diagnosis, efficacy evaluation, and prognosis of diseases using CK17 protein as a biomarker, such as epithelial tumors and skin diseases, which is beneficial for disease early warning, screening, intervention, and efficacy evaluation. Therefore, this invention provides more protein options for the detection of CK17 protein and for early warning, diagnosis, efficacy evaluation, and prognosis of epithelial tumors and skin diseases.

[0050] In a preferred embodiment of the present invention, the above-mentioned binding protein binds to the CK17 antigen at a K... D ≤2.228×10 8 The affinity binding is L / mol, and the bound protein is an antibody or a functional fragment. K D The detection is performed in accordance with the method described in the embodiments of the present invention.

[0051] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0052] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes the following frame region:

[0053] The HFR1 amino acid sequence has at least 80% homology with SEQ ID NO:7;

[0054] The HFR2 amino acid sequence has at least 80% homology with the one shown in SEQ ID NO:8;

[0055] The HFR3 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:9;

[0056] The HFR4 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:10;

[0057] The LFR1 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:11;

[0058] The LFR2 amino acid sequence has at least 80% homology with the one shown in SEQ ID NO:12;

[0059] The LFR3 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:13;

[0060] The LFR4 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:14.

[0061]

[0062] It should be noted that, in other embodiments, the amino acid sequences of each backbone region of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0063] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region;

[0064] In a preferred embodiment of the present invention, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0065] In a preferred embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0066] In a preferred embodiment of the present invention, the heavy chain constant region (CH) sequence is as shown in SEQ ID NO:15 or has at least 80% homology with it, and the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% homology with it.

[0067] In other embodiments, the heavy chain constant region amino acid sequence of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the above-mentioned SEQ ID NO:15.

[0068] In other embodiments, the light chain constant region (CL) amino acid sequence of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the above-mentioned SEQ ID NO:16.

[0069] In a preferred embodiment of the present invention, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0070] In a preferred embodiment of the present invention, the species source of the constant region is mouse or human;

[0071] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of the antibody F(ab')2, Fab', Fab, Fv and scFv.

[0072] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0073] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0074] Secondly, the present invention also provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0075] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is labeled with a detectable marker; the detectable marker refers to a substance that has properties that can be directly observed by the naked eye or detected or probed by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0076] In a preferred embodiment of the present invention, the detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers. In practical use, those skilled in the art can select appropriate markers based on detection conditions or actual needs; regardless of the marker used, it falls within the scope of protection of this invention.

[0077] 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.).

[0078] 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.

[0079] 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.

[0080] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0081] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0082] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0083] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0084] In a preferred embodiment of the present invention, CK17 protein detection is performed by binding protein-labeled tissue cells.

[0085] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is coated onto a solid phase; for example, the antibody or its antigen-binding fragment is connected to the solid phase by chemical coupling.

[0086] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates, and membranes;

[0087] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0088] Thirdly, the present invention also provides the use of the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate in the preparation of any of the following products, wherein the product is selected from:

[0089] Reagents, kits, and chips.

[0090] In a preferred embodiment of the present invention, the product has at least one of the following uses: for risk warning, diagnosis, efficacy monitoring, prognostic assessment, CK17 protein detection, CK17 protein enrichment, and CK17 protein purification of tumor or non-tumor skin diseases.

[0091] In a preferred embodiment of the present invention, the tumor is selected from epithelial tumors;

[0092] In a preferred embodiment of the present invention, the epithelial tumor is selected from papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, melanoma, basal cell carcinoma, or squamous cell carcinoma.

[0093] 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.

[0094] In one alternative implementation, adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0095] In one alternative implementation, the adenoma includes, but is not limited to, cystic adenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma.

[0096] In one alternative implementation, breast cancer includes, but is not limited to, breast epithelial carcinoma;

[0097] Preferably, the breast cancer is ductal carcinoma or lobular carcinoma of the breast.

[0098] Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma and medullary carcinoma (preferably grade 2).

[0099] Ovarian cancer, serous and mucinous carcinoma (preferably stage Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0100] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0101] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0102] 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.

[0103] In a preferred embodiment of the present invention, squamous cell carcinoma is selected from one or more of the following: lung squamous cell carcinoma, cervical squamous cell carcinoma, tonsil carcinoma, oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal carcinoma, esophageal squamous cell carcinoma, squamous cell carcinoma of the lip, and squamous cell carcinoma of the skin.

[0104] 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.

[0105] 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.

[0106] In a preferred embodiment of the present invention, the non-tumor skin disease is selected from psoriasis, dermatitis, hidradenitis suppurativa, oral lichen planus, chronic herpes disease, acne, seborrheic dermatitis of immune-mediated diseases, alopecia areata, alopecia areata, sclerema, scarring, urticaria, rosacea, or inflammation originating from kidney transplantation.

[0107] In a preferred embodiment of the present invention, the dermatitis is selected from exfoliative dermatitis, pityriasis rosea, lichen planus, lichen luster, porokeratosis, or seborrheic keratosis.

[0108] In some implementations, the aforementioned non-neoplastic skin diseases include skin diseases characterized by persistent inflammation, active cells, and differentiation (e.g., psoriasis, psoriatic arthritis, exfoliative dermatitis, pityriasis rosea, lichen planus, lichen luster, or porokeratosis); skin diseases characterized by epidermal adhesions, vesicular and bullous disorders (e.g., pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, or pustular eczema of the palms or soles); skin diseases and related conditions of epidermal appendages (e.g., trichoderma, nail diseases, rosacea, perioral dermatitis, or folliculitis syndrome); melanocyte disorders (e.g., pigmentary disorders, albinism, hypomelanosis and hypermelanosis, melanocytic nevus); and dermatological diseases of inflammatory dermis (e.g., persistent erythema raisedae, eosinophilic dermatitis, pyoderma gangrenosa, malignant atrophy). Petechiae, fibrotic lesions of the dermis and soft tissues; subcutaneous tissue diseases (e.g., panniculitis or lipodystrophy); dermatitis involving altered reactive skin changes (e.g., urticaria, angioedema, graft-versus-host disease, allergic contact dermatitis, autosensitization dermatitis, atopic dermatitis, or seborrheic dermatitis); skin changes due to mechanical and physical factors (e.g., heat injury, radiation dermatitis, corns, or calluses); photodamage (e.g., acute and chronic UV radiation, or photosensitivity); or dermatitis caused by microbial agents (e.g., leprosy, Lyme spirochete, onychomycosis, tinea pedis, rubella, measles, herpes simplex, EBV (Epstein-Barr virus), HPV (human papillomavirus) (e.g., HPV6 & 7), warts, or prions).

[0109] Applications of CK17 protein enrichment and purification include, but are not limited to: coating the above-mentioned antibody or antigen-binding fragments onto a solid phase (such as setting them on an adsorption column through chemical modification), and achieving the separation, enrichment and purification of CK17 protein in the sample through affinity chromatography and elution.

[0110] Fourthly, the present invention also provides a reagent or kit for risk warning, diagnosis, efficacy monitoring, prognostic assessment, CK17 protein detection, CK17 protein enrichment, or CK17 protein purification of tumors or non-tumor skin diseases, which contains the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0111] Fifthly, the present invention also provides a nucleic acid that encodes the aforementioned antibody or its antigen-binding fragment.

[0112] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; 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.

[0113] In a sixth aspect, the present invention also provides a carrier comprising the aforementioned nucleic acid.

[0114] The 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.

[0115] In a seventh aspect, the present invention also provides a cell comprising the aforementioned carrier.

[0116] The cells are selected from mammalian cells; the mammalian cells are selected from any one of 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.

[0117] Eighthly, the present invention also provides a method for producing the aforementioned antibody or its antigen-binding fragment, comprising:

[0118] The cells described in the above embodiments are cultured, and the binding protein is isolated and purified from the culture medium or from the cultured cells.

[0119] The production method may, for example, involve transfecting host cells with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cells under suitable conditions to express the binding protein. The host cells may also be transfected with one or more expression vectors, which may contain DNA encoding at least a portion of the binding protein, either alone or in combination. The binding protein can be isolated from culture medium or cell lysates using conventional protein and peptide purification techniques, including ammonium sulfate precipitation, chromatography (such as ion exchange, gel filtration, affinity chromatography, etc.), and / or electrophoresis.

[0120] Constructing suitable vectors containing the target coding and regulatory sequences can be performed using standard ligation and restriction techniques known in the art. Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cut, tailed, and re-ligated as needed. Mutations can be introduced into the coding sequence using any method to produce variants of the invention; these mutations can include deletions, insertions, substitutions, etc.

[0121] 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.

[0122] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0123] Example 1

[0124] This embodiment describes the preparation of a monoclonal antibody specifically targeting the CK17 protein.

[0125] 1. Animal (mouse) immunization

[0126] Mice were immunized with an immunogen using a standard method. The immunogen was human CK17 protein, which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of CK17 antigen. The antigen-protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the syringe was placed on an emulsifier and stirred thoroughly to form a stable water-in-oil solution. The first tail blood serum titer was measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for final intraperitoneal immunization followed by cell fusion.

[0127] 2. Hybridoma cell fusion and screening

[0128] Preparations are required before cell fusion: (1) Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; (2) Sacrifice a negative mouse one day before fusion, and inject mouse peritoneal trophoblast cells into the peritoneal cavity in a sterile environment using HAT medium to collect the cells and plate them in 96-well plates, 100 μL per well. These cells promote the growth of hybridoma cells. Sacrifice the immunized mouse, and collect the spleen in a sterile environment. Use PEG to chemically fuse spleen B cells and SP2 / 0 myeloma cells. Add appropriate HAT medium according to the number of cells to be plated, and finally plate the fused cells in a trophoblast cell culture plate, 100 μL per well.

[0129] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant from each well is collected, and hybridoma cells are screened using the human ck17-his protein antigen via ELISA.

[0130] The method is as follows: Coat the ELISA plate with 100 μL of PBS solution containing 2 μg / ml human ck17 protein antigen and incubate at 37°C for two hours. After washing the plate three times with PBST, add 150 μL / well of PBS solution containing 3% skim milk powder and incubate overnight at 4°C. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:8000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, develop at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0131] Example 2

[0132] The ELISA-positive fusion wells from Example 1 were selected for immunohistochemical (IHC) experiments. The experimental steps are as follows:

[0133] Lung squamous cell carcinoma was baked in a 60℃ constant temperature oven for 60 minutes. The slides were soaked in xylene I for 15 minutes, then in xylene II for 15 minutes. They were then soaked in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and ddH2O for 5 minutes. The slides were washed 3 times. Antigen retrieval was performed using a pressure cooker (boiling method). 10 mmol / L citrate buffer (pH 6.0) was added to the pressure cooker to submerge the slides. The pressure was heated to boiling. The slides were placed on a heat-resistant slide rack and placed in the pressure cooker. The lid was closed, the pressure valve was closed, and the pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the pressure. After the pressure returned to zero, the lid was opened, the inner pot was removed, and the pressure was allowed to cool to room temperature. After the solution cools to room temperature, remove the sections (about 40 min); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash 3 times; add one drop of goat serum blocking solution (about 25 μl) to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash 3 times.

[0134] The processed tissue sections were added to Maixin's CK17 antibody (CAT#:MAB-2673) for comparison, while the remaining sections were added to the ELISA-positive fusion wells purified antibody from Example 1. Incubate overnight in a humidified chamber at 4°C; remove from the refrigerator at 4°C and incubate at room temperature for 60 minutes; gently rinse with PBST and soak for 5 minutes, washing 3 times; add 25 μL of HRP-labeled Long Island Biotechnology secondary antibody (D-3004-0100) to each tissue group and incubate at room temperature for 45 minutes; wash; prepare DAB staining solution, react in the dark for 10-15 minutes, then drop onto the sections and develop for 1-5 minutes; terminate the staining reaction with distilled water; add 50 μL of hematoxylin staining solution to each tissue group and stain for 5-10 minutes, then rinse thoroughly with distilled water; decolorize the sections in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove and place in distilled water to terminate the staining, then place in PBST (pH 8.0) for 5-10 minutes for inversion; soak in 75% ethanol for 5 minutes; soak in 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin to seal the slide, then cover with a coverslip; take a microscope image.

[0135] Fusion cells that showed positive binding were selected using the ELISA assay in Example 1 and the IHC assay in Example 2. These cells were then cloned using the limiting dilution method, with each positive cell line seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA to identify hybridomas that specifically recognize the ck17 protein and can block CK17 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 1B2.

[0136] The monoclonal cell line was expanded, and approximately 1 × 10⁻⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against CK17 protein (CK17-1B2 antibody) was obtained.

[0137] Figure 1 shows the immunohistochemical results of the section with CK17-1B2 antibody added. The specimen tested in Figure 1 is tonsil tissue. As can be seen from the staining results in Figure 1, there is no significant difference in the staining sites and staining intensity between CK17-1B2 antibody and Maixin antibody. This confirms that the CK17 provided by this invention has promising application prospects in the preparation of kits.

[0138] Example 3

[0139] DNA cloning and sequencing were performed, including sequencing of the variable region gene of the anti-human CK17 monoclonal antibody.

[0140] Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of Trizol reagent was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 minutes to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds, then incubated at room temperature for 3 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The mixture was centrifuged at 12000 rpm at 4°C for 3 minutes, and the supernatant was discarded. The mixture was dried at room temperature for 5-10 minutes. 30-50ul of RNase-free ddH2O was added. The resulting RNA solution was stored at -70°C or used for subsequent experiments.

[0141] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While in the ice bath, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds, and then incubated on a PCR instrument at 42°C for 50 minutes and 85°C for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

[0142] PCR amplification of the light and heavy chains. For amplifying the variable region sequence of the antibody light chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VL + 1 μL RP-VL + 2 μL cDNA + 21 μL ddH2O. For amplifying the variable region sequence of the antibody heavy chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VH + 1 μL RP-VH + 2 μL cDNA + 21 μL ddH2O. The temperature cycling for PCR amplification of the variable regions of the heavy and light chains was as follows (steps 2 to 4 were repeated 35 times):

[0143] Step 1 - Pre-denaturation: 94℃, 4 min;

[0144] Step 2 - Denaturation at 94°C for 30 seconds;

[0145] Step 3 - Annealing at 55°C for 45 seconds;

[0146] Step 4 - Extend at 72°C for 60 seconds;

[0147] Step 5: 72℃, 10 min;

[0148] Step 6 - Store at 4℃.

[0149] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 375 bp for VH and approximately 325 bp for VL). DNA extraction was performed using the SanPrep DNA Gel Extraction Kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was incubated at 50°C for 5-10 minutes to dissolve; the solution was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the adsorption column was centrifuged at 9000g for 1 minute; the adsorption column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was purified and sequenced to obtain the variable region sequence of the antibody.

[0150] Experimental Example 1

[0151] This experimental example tests the affinity and sensitivity of the antibody (CK17-1B2 purified antibody) prepared in Example 2 above.

[0152] 1. Recombinant human CK17 protein (SEQ ID NO.17) was plated at concentrations of 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L, respectively.

[0153] 2. Adjust the antibody concentration to 10. -7 mol / L level (1*10) -7 Up to 5*10 -7 (Mol / L is acceptable). Then serially dilute 1:2 to 1:256 and add to wells with different antigen coating amounts.

[0154] 3. Add Sangon Biotech's secondary antibody D110065 and perform TMB colorimetric assay. Measure the absorbance at 450 nm; the data are shown in Table 1.

[0155] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This will result in four antibody concentrations (mol / L).

[0156] 5. Substitute the values ​​into the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB' and AB are the antibody concentrations that produce the half-maximum absorbance at the corresponding antigen concentrations AG (3 mg / L, 1.5 ml / L, 0.75 mg / L, 0.375 mg / L). N = AG / AG' (AG > AG').

[0157] 6. When N=2, we get three K values: 0.556, 0.177, and 0.109. When N=4, we get two K values: 0.230 and 0.1725. When N=8, we get one K value: 0.140. The average of the six K values ​​is 2.228 × 10⁻⁶. 8 L / mol.

[0158] Table 1. Statistical Table of Absorbance Values ​​at 450nm

[0159]

[0160] 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 against CK17 or its antigen-binding fragment, characterized in that, The antibody comprises the following complementarity-determining regions: HCDR1, consisting of the amino acid sequence shown in SEQ ID NO:1; HCDR2, consisting of the amino acid sequence shown in SEQ ID NO:2; HCDR3, consisting of the amino acid sequence shown in SEQ ID NO:3; LCDR1, consisting of the amino acid sequence shown in SEQ ID NO:4; LCDR2, consisting of the amino acid sequence shown in SEQ ID NO:5; and LCDR3, consisting of the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-CK17 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes the following frame regions: the HFR1 amino acid sequence has at least 80% homology with SEQ ID NO:7; the HFR2 amino acid sequence has at least 80% homology with SEQ ID NO:8; the HFR3 amino acid sequence has at least 80% homology with SEQ ID NO:9; the HFR4 amino acid sequence has at least 80% homology with SEQ ID NO:10; the LFR1 amino acid sequence has at least 80% homology with SEQ ID NO:11; the LFR2 amino acid sequence has at least 80% homology with SEQ ID NO:12; the LFR3 amino acid sequence has at least 80% homology with SEQ ID NO:13; and the LFR4 amino acid sequence has at least 80% homology with SEQ ID NO:

14.

3. The anti-CK17 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment also includes a constant region.

4. The anti-CK17 antibody or its antigen-binding fragment according to claim 3, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

5. The anti-CK17 antibody or its antigen-binding fragment according to claim 4, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

6. The anti-CK17 antibody or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% homology with it, and the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% homology with it.

7. The anti-CK17 antibody or its antigen-binding fragment according to claim 3, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

8. The anti-CK17 antibody or its antigen-binding fragment according to claim 3, characterized in that, The species source of the constant region is mice or humans.

9. The antibody against CK17 or its antigen-binding fragment according to any one of claims 1-8, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

10. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof is labeled with a detectable marker.

11. The antibody conjugate according to claim 10, characterized in that, The detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

12. The antibody conjugate according to claim 11, characterized in that, The antibody or its antigen-binding fragment is coated in a solid phase; the solid phase is selected from microspheres, plates, and membranes.

13. The antibody conjugate according to claim 11, characterized in that, The solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

14. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, or the antibody-drug conjugate as described in any one of claims 10-13, in the preparation of any of the following products, characterized in that, The product is selected from reagents, kits, and chips; the product is used for CK17 protein detection.

15. A reagent or kit for detecting CK17 protein, characterized in that, It contains the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 or the antibody conjugate as described in any one of claims 10-13.

16. A nucleic acid, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

17. A carrier, characterized in that, It includes the nucleic acid as described in claim 16.

18. A cell characterized in that, It includes the carrier as described in claim 17.

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