Antibody specifically targeting CLDN6 and application thereof in preparation of CAR-T cells

Abstract

The invention provides an antibody specifically targeting CLDN6 and an application of the antibody in preparation of CAR-T cells. The CLDN6 antibody provided by the invention specifically targets CLDN6 and does not recognize CLDN3, CLDN4 and CLDN9, or the recognition modes are obviously different. An antibody sequence based on specific targeting CLDN6 can be used for constructing a chimeric antigen receptor (CAR) and a bivalent antibody (bispecific antibody). The CAR constructed based on the antibody sequence can be transduced into a T cell to create a CAR-T cell specifically targeting CLDN6, and the CAR-T cell can be used for treating solid tumors such as ovarian cancer.

Description

Technical Field

[0001] The present invention relates to an antibody specifically targeting CLDN6 and an application thereof in preparing CAR-T cells, and relates to the fields of genetic engineering and antibody technology. Background Art

[0002] CLDN6 (Claudin-6) is a tumor-specific protein that is expressed in a variety of solid tumors, including ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer, but is almost not expressed in healthy adult tissues. This specific expression pattern makes CLDN6 a very promising target for tumor therapy.

[0003] CLDN6 belongs to the claudin protein family, which has 24 members expressed in mammals. Among them, CLDN3 is widely expressed in a variety of epithelial tissues, including the transverse colon mucosa and gastrointestinal tract of the digestive system, the endometrial epithelium and right fallopian tube of the reproductive system, as well as a variety of glandular tissues and type II alveolar epithelial cells. CLDN4 has a wider range of expression, with the most abundant expression in the gastrointestinal tract, and is also expressed in adipose tissue, adrenal glands, tonsils, appendix, basal ganglia, bone marrow, breast, bronchi, cerebellum, cerebral cortex, cervix, choroid plexus, colon, duodenum, endometrium and other tissues. In contrast, CLDN9 is more limited in expression and mainly plays an important role in the cochlea of ​​the inner ear, participating in the regulation of ion balance and hearing function of the inner ear. CLDN6 has a high structural homology with CLDN3, CLDN4 and CLDN9, especially the extracellular domains of CLDN6 and CLDN9 have only 3 amino acid differences. The widespread expression of these family proteins also affects the safety of the use of CLDN6 antibodies.

[0004] Based on the above background, there is an urgent need for an antibody that can specifically target CLDN6 without binding to CLDN3, CLDN4 and CLDN9, so as to improve the safety of antibody use while not affecting other normal physiological functions of the claudin protein family during tumor treatment. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an antibody that specifically targets CLDN6 but does not bind to CLDN3, CLDN4 and CLDN9. Specifically, the present invention includes the following contents:

[0006] The first aspect of the present invention provides an antibody specifically targeting CLDN6, the antibody comprising:

[0007] 1) HCDR1-HCDR3 of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4; or 2) HCDR1-HCDR3 of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or 3) HCDR1-HCDR3 of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12; or 4) HCDR1-HCDR3 of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:16; or 5) HCDR1-HCDR3 of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:20.

[0008] Furthermore, the numbering schemes for the CDRs include IMGT, Chothia, Kabat, AbM, and Contact.

[0009] In certain embodiments, the sequence of HCDR1-3 of the heavy chain variable region of the antibody described in the present invention can be obtained based on the full-length sequence of the above-mentioned heavy chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequence defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system is also within the scope of protection of the present invention.

[0010] Furthermore, the numbering scheme of the CDR is IMGT.

[0011] Further, the sequence of HCDR1-HCDR3 of the heavy chain variable region includes: the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO:1-3; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO:5-7; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO:9-11; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO:13-15; or the amino acid sequence of the HCDR1-HCDR3 is shown in SEQ ID NO:17-19.

[0012] Furthermore, the antibody also includes a heavy chain variable region in which part of the amino acids in the framework region of 4) or 5) are replaced with humanized amino acids.

[0013] In certain embodiments, "humanization" described in the present invention generally refers to humanizing non-human antibodies to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, the CDR (or part thereof) of a humanized antibody is derived from a non-human antibody sequence, and the FR (or part thereof) is derived from a human antibody sequence. Optionally, a humanized antibody also includes at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0014] Furthermore, the heavy chain variable region in which some amino acids in the framework region of 4) are replaced with humanized amino acids is as shown in any of the following groups: a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21; b) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:22; c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23; d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:24.

[0015] Further, the heavy chain variable region in which some amino acids in the framework region of 5) are replaced with humanized amino acids is as shown in any of the following groups: e) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25; f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27; h) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28; i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29; j) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:30; k) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31.

[0016] Furthermore, the antibody also includes an Fc sequence.

[0017] Furthermore, the Fc sequence is shown in SEQ ID NO:32.

[0018] Furthermore, the antibody also includes an export signal peptide.

[0019] Furthermore, the sequence of the membrane export signal peptide is shown in SEQ ID NO:33.

[0020] Furthermore, the antibody includes a single domain antibody, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody or a bivalent antibody.

[0021] Furthermore, the antibody is a single domain antibody.

[0022] The second aspect of the present invention provides an antibody derivative, wherein the antibody derivative comprises any one of the following:

[0023] a. an antibody-marker conjugate, wherein the antibody-marker conjugate comprises the antibody according to the first aspect of the present invention and a detectable marker conjugated thereto;

[0024] b. a pharmaceutical composition comprising the antibody according to the first aspect of the present invention and a pharmaceutically acceptable carrier;

[0025] c. A chimeric antigen receptor comprising the heavy chain variable region of the antibody according to the first aspect of the present invention.

[0026] Furthermore, the detectable marker comprises at least one of a radioactive isotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0027] Furthermore, the chimeric antigen receptor also includes one or more of an extracellular hinge region, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory domain.

[0028] Further, the extracellular hinge region is selected from the extracellular hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.

[0029] Furthermore, the extracellular hinge region is CD8 Hinge.

[0030] Furthermore, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO:34.

[0031] Further, the transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.

[0032] Furthermore, the transmembrane domain is CD8 TM.

[0033] Furthermore, the amino acid sequence of the CD8 TM is shown in SEQ ID NO:35.

[0034] Further, the intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and their variants.

[0035] Furthermore, the intracellular signaling domain is the CD3ζ intracellular signaling domain.

[0036] Furthermore, the amino acid sequence of CD3ζ is shown in SEQ ID NO:36.

[0037] Furthermore, the chimeric antigen receptor also includes a co-stimulatory signaling domain.

[0038] Further, the costimulatory domain is selected from the costimulatory domains of the following molecules: 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof.

[0039] Furthermore, the co-stimulatory signaling domain is 4-1BB.

[0040] Furthermore, the amino acid sequence of the 4-1BB is shown in SEQ ID NO:37.

[0041] Furthermore, the chimeric antigen receptor also includes a suicide gene or a detectable tag.

[0042] Furthermore, the suicide gene or detectable tag includes EGFRt.

[0043] Furthermore, the amino acid sequence of the EGFRt is shown in SEQ ID NO:38.

[0044] Furthermore, the suicide gene or detectable tag also includes an EGFRt membrane exit signal.

[0045] Furthermore, the amino acid sequence of the EGFRt export signal is shown in SEQ ID NO:39.

[0046] Furthermore, the chimeric antigen receptor also includes a linker.

[0047] Furthermore, the linker includes 2A peptide and IRES.

[0048] Furthermore, the 2A peptides include P2A, T2A, E2A and F2A.

[0049] Furthermore, the linker is a 2A peptide.

[0050] Furthermore, the 2A peptide is T2A.

[0051] Furthermore, the amino acid sequence of T2A is shown in SEQ ID NO:40.

[0052] Furthermore, the chimeric antigen receptor also includes a signal peptide.

[0053] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO:41.

[0054] In certain embodiments, those skilled in the art can change the combination category and sequence of the signal peptide, the extracellular hinge region and the transmembrane domain, the co-stimulatory domain and the intracellular signal domain according to actual conditions or needs. No matter what form of change is based on, as long as the chimeric antigen receptor has the CDR sequence or the heavy chain variable region sequence of the heavy chain variable region of the above-mentioned humanized antibody of the present invention, it belongs to the protection scope of the present invention. Most preferably, the chimeric antigen receptor is selected from any one of the following groups: 1. A chimeric antigen receptor obtained by sequentially connecting the signal peptide, the antibody described in the first aspect of the present invention, CD8 Hinge, CD8 TM, 4 1BB co-stimulatory domain, CD3ζ intracellular signal domain, T2A, EGFRt signal peptide, and EGFRt in series; 2. A derivative chimeric antigen receptor formed by replacing, deleting or adding one or more amino acids based on the amino acid sequence of the chimeric antigen receptor described in 1.

[0055] The third aspect of the present invention provides a biomaterial comprising:

[0056] (I) a nucleic acid molecule encoding the antibody of the first aspect of the present invention or the antibody derivative of the second aspect of the present invention; or

[0057] (II) a vector comprising the nucleic acid molecule described in (I); or

[0058] (III) a recombinant host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II); or

[0059] (IV) A CAR-T cell comprising the chimeric antigen receptor described in the second aspect of the present invention.

[0060] The recombinant host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. Host cells include any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cell includes bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, rickettsia; more preferably, the eukaryotic cell includes mammalian cells, insect cells, plant cells, yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cell is a T cell.

[0061] Furthermore, the nucleic acid molecule comprises a base sequence encoding HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:43-58.

[0062] Furthermore, the base sequences encoding HCDR1-HCDR3 are shown in SEQ ID NOs: 59-73.

[0063] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO:74.

[0064] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO:75.

[0065] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO:76.

[0066] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD8 TM as shown in SEQ ID NO:77.

[0067] Furthermore, the nucleic acid molecule also comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:78.

[0068] Furthermore, the nucleic acid molecule also comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:79.

[0069] Furthermore, the nucleic acid molecule also comprises a base sequence encoding EGFRt as shown in SEQ ID NO:80.

[0070] Furthermore, the nucleic acid molecule also comprises a base sequence encoding an export signal of EGFRt as shown in SEQ ID NO:81.

[0071] Furthermore, the nucleic acid molecule also comprises a base sequence encoding T2A as shown in SEQ ID NO:82.

[0072] Furthermore, the nucleic acid molecule also comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO:83.

[0073] The fourth aspect of the present invention provides the use of the antibody according to the first aspect of the present invention, the chimeric antigen receptor in the antibody derivative according to the second aspect of the present invention, and / or the nucleic acid molecule in the biomaterial according to the third aspect of the present invention, wherein the use includes any of the following:

[0074] Application in the preparation of products for detecting CLDN6 protein.

[0075] Application in constructing a bivalent antibody targeting CLDN6.

[0076] Application in constructing immune cells that specifically target CLDN6.

[0077] Application in the treatment of CLDN6-positive related diseases.

[0078] Application in the preparation of products for treating CLDN6-positive related diseases.

[0079] Furthermore, the products for treating CLDN6-positive related diseases include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips.

[0080] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0081] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0082] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0083] Furthermore, the ovarian epithelial tumor includes an ovarian adenocarcinoma tumor.

[0084] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0085] A fifth aspect of the present invention provides a method, the method comprising any of the following:

[0086] (1) An in vitro method for detecting CLDN6 in a sample for non-therapeutic purposes, the method comprising: contacting the sample with the antibody of the first aspect of the present invention or the antibody-marker conjugate in the composition of the second aspect of the present invention, and detecting the formation of a complex between the antibody and CLDN6.

[0087] (2) A method for producing the antibody of the first aspect of the present invention, the method comprising: culturing the recombinant host cell in the biological material of the third aspect of the present invention, and isolating the antibody of the first aspect of the present invention from the culture.

[0088] (3) An in vitro method for promoting apoptosis of ovarian adenocarcinoma cells for non-therapeutic purposes, the method comprising co-culturing the CAR-T cells in the biomaterial described in the third aspect of the present invention with ovarian adenocarcinoma cells.

[0089] Furthermore, the ovarian adenocarcinoma cell is SKOV3.

[0090] Another aspect of the present invention provides a method or pharmaceutical composition for treating a CLDN6-positive disease in a subject, the method comprising administering to the subject the antibody of the first aspect of the present invention, the antibody derivative of the second aspect of the present invention, and / or the CAR-T cells in the biomaterial of the third aspect of the present invention. In some embodiments, the subject comprises a mammal, and in a specific embodiment of the present invention, the subject is preferably a human.

[0091] Furthermore, the CLDN6-positive related diseases include solid tumors.

[0092] Furthermore, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer.

[0093] Furthermore, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors.

[0094] Furthermore, the ovarian epithelial tumor includes an ovarian adenocarcinoma tumor.

[0095] Furthermore, the solid tumor is an ovarian adenocarcinoma tumor.

[0096] Furthermore, the pharmaceutical composition comprises an effective amount of the antibody described in the first aspect of the present invention, the antibody derivative described in the second aspect of the present invention and / or the CAR-T cells in the biomaterial described in the third aspect of the present invention and a pharmaceutically acceptable carrier.

[0097] The term "effective amount" herein relates to an amount of an active compound or a material, composition or dosage form comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0098] Advantages and beneficial effects of the present invention: The CLDN6 antibody provided by the present invention specifically targets CLDN6, but does not recognize CLDN3, CLDN4, CLDN9, or has obvious differences in recognition mode. Antibody sequences based on specific targeting of CLDN6 can be used to construct chimeric antigen receptors (CAR) and bivalent antibodies (bispecific antibodies). CARs constructed based on the antibody sequences can be transduced into T cells to create CAR-T cells that specifically target CLDN6, which can be used to treat solid tumors such as ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 Diagram of the construction of a phage display library for alpaca immunization;

[0100] Figure 2 This is a diagram showing the FACS binding validation results of CLDN6 transfection supernatant;

[0101] Figure 3 This is a diagram showing the validation results of the candidate antibodies binding to cells expressing CLDN6-related homologous proteins;

[0102] Figure 4 This is a diagram showing the results of binding validation of purified candidate antibodies to CLDN6 homologous proteins;

[0103] Figure 5 This is a graph showing the results of affinity analysis of CLDN6 antibodies;

[0104] Figure 6 This is the FACS result of 2D11 humanized sequence;

[0105] Figure 7 This is a graph showing the affinity test results of 2D11 humanized antibody;

[0106] Figure 8 This is the FACS result of 1H07 humanized sequence;

[0107] Fig. 9 This is the affinity result diagram of 1H07 humanized sequence;

[0108] Fig.10 Schematic diagram for the construction of CAR-T vector;

[0109] Fig.11 This is a graph showing the killing rate of CLDN6-2D11 and humanized antibody 2D11-HM7 CAR-T;

[0110] Fig.12 The figure shows the killing rate of CLDN6-2D11, humanized 2D11-HM4, 1H07 and humanized 1H07-HM2 CAR-T cells;

[0111] Fig.13This is a graph showing the killing rate of candidate antibodies against SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9. DETAILED DESCRIPTION

[0112] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0113] In the context of the present invention, the term "antibody" is used in the broadest sense and specifically covers single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0114] In the present invention, the term "single-domain antibodies" (sdAbs), also known as VHH antibodies or camelid antibodies, is an artificially designed antibody molecule, which is a natural light chain-deficient heavy-chain antibody (HCAbs) found in camelids such as alpacas and dromedaries, and cartilaginous fish such as sharks and rays, including two constant regions (CH2 and CH3), a hinge region and a variable heavy chain domain (VHH), and then cloned to obtain a single-domain antibody containing only one heavy chain variable region, namely, a VHH antibody. The crystal structure of VHH antibody is an ellipse of 4nm×2.5nm×3nm, and the molecular weight is only 1 / 10 of that of ordinary antibodies, about 12-14kDa, which is the smallest complete antigen-binding fragment, so it is also called a nanobody. The term "antigen-binding fragment" generally refers to one or more fragments of an antibody that play a specific antigen-binding function.

[0115] In the present invention, the terms "specificity", "binding" and "targeting" refer to that the binding effect is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art. In a specific embodiment of the present invention, specificity and targeting refer to that the antibody described in the first aspect of the present invention can bind to CLDN6, but not to CLDN3 / 4 / 9, or the binding mode is significantly different so that it can be significantly distinguished from CLDN6.

[0116] In the present invention, the antibody sequence obtained by modification and having 80% or more identity with the amino acid sequence of the antibody described in the first aspect of the present invention also belongs to the protection scope of the present invention. The term "modification" refers to any form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to the addition of one or more amino acids compared to the original amino acid sequence caused by the change in the amino acid sequence. The term "identity", also known as "homology", refers to an amino acid sequence whose sequence is at least 80% identical to the sequence provided by the present invention. In order to determine the sequence identity, sequence alignment can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software, etc. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithm required to achieve optimal alignment in the full-length sequence being compared.

[0117] Unless otherwise indicated, the antibodies described herein are isolated antibodies. The term "isolated" as used herein refers to a nucleic acid or antibody or fragment thereof that has been extracted from its natural environment. Nucleic acids or antibodies or fragments thereof that have been "isolated" thus include nucleic acids or antibodies or fragments thereof purified by standard purification methods. The term also includes nucleic acids or antibodies or fragments thereof prepared by recombinant expression in a host cell and chemically synthesized nucleic acids and / or antibodies.

[0118] In the present invention, the term "nucleic acid" or "nucleic acid molecule" is intended to include polymeric forms of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, which also include DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into a cell, and then isolate it from the host cell after proliferation by conventional methods.

[0119] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention or a precursor thereof, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0120] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention are obtained by direct amino acid synthesis. In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Among them, prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0121] In the present invention, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implanted drug storage devices. The pharmaceutical composition of the present invention can contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant, or excipient. In some cases, pharmaceutical acids, bases, or buffers can be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the receptor by any route.

[0122] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating solid tumors. Other compounds for treating solid tumors can be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the present invention), or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient.

[0123] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0124] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials and reagents used in the following examples, unless otherwise specified, are obtained from commercial sources. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0125] Example 1 Construction of phage display library, antibody selection and humanization

[0126] 1. Alpaca immunization to construct phage display library

[0127] Two alpacas were immunized with 293F-CLDN6, with an interval of 14 days. Seven days after the last immunization, some peripheral blood was collected, and the serum was separated and tested for immune titer using ELISA. After passing the test, PBMC was separated using lymphocyte separation solution. RNA was extracted using PrimeScript TM II 1st Strand cDNA Synthesis Kit was used for reverse transcription to prepare cDNA. The single-domain antibody cloning primer combination was used to amplify the VHH sequence from the cDNA sample and subcloned into the phage display vector pDisplay. The SS320 competent cells were electroporated to construct a single-domain antibody phage display library. The library was constructed by mixing two alpacas, sequencing 23 clones, removing two antibody sequences with one base missing and one overlapping peak. The remaining sequences were all antibody sequences with good diversity. The phage display library was as follows Figure 1 shown.

[0128] 2. Select candidate antibody clones for specific recognition verification

[0129] CLDN6-VLP and CHO-S-Lenti-CMV-hCLDN6 cells were used for panning, and CHO-S-Lenti-CMV-hCLDN6 cells were used for negative screening. According to the phage ELISA test, 8 different antibody sequences were obtained by solid phase and cell panning. After PCR, SfiI restriction enzyme digestion and subcloning into pcDNA3.4-IgG1Fc expression vector were used for CLDN6 transfection supernatant FACS binding verification experiment. The experiment used 3×10 5 Each well corresponds to overexpression or blank control cells, the primary antibody is CLDN6 target transfection supernatant (100 μl / well), and the secondary antibody is PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:500 dilution). Among them, PC is the positive control (clone number: AB89A, reference patent: CN111875703A). The experimental results are shown in Figure 2 As shown, it can be seen that candidate clones 1-D05, 1-G01, 1-H06, 1-H07, 2-D11 and 3-D12 bind more strongly to CHO-S-CLDN6 cells.

[0130] The expression, purification and detection of 5 candidate antibodies were used to verify the binding of antibodies to cells overexpressing CLDN3 / 4 / 6 / 9. CHO-S control cells and 3×10 CHO-S cells overexpressing CLDN3 / 4 / 6 / 9 were used. 5 / well, CLDN6 target candidate antibody (10μg / ml, 100μl / well) was used as the primary antibody for incubation at room temperature for 1 hour, and after washing the cells 3 times with PBS, PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:500 dilution) was used as the secondary antibody for incubation at room temperature in the dark for 45 minutes, and after washing the cells again with PBS 3 times, the cells were resuspended in 200μL PBS for flow cytometry analysis. The results showed that the four purified antibodies 1-D05, 1-G01, 1-H06 and 2-D11 basically did not bind to CLDN3 and CLDN4, and 1-H07 bound strongly to CLDN4, but had obvious differences from CLDN6. The three antibodies 2-D11, 1-H07, and 8-G02 were selected for further FACS binding detection (the concentrations of the candidate antibodies for the CLDN6 target were 30 μg / ml, 10 μg / ml, and 3 μg / ml, 100 μl / well). The results are as follows Figure 3 As shown, 1-H07 and 2-D11 were found to be ideal. Among them, PC (731B2) is an antibody from US11345731 patent.

[0131] 3. Affinity verification of candidate antibodies

[0132] After all candidate antibodies were purified, they were tested with gradient dilutions of cells overexpressing CLDN6 and CLDN9 (CLDN6 target candidate antibodies were diluted 3 times to 10 points starting from 30 μg / ml). The results are as follows: Figure 4 , Figure 5 As shown, only 1-H07 and 2-D11 basically did not bind to CLDN9, and the remaining antibodies all bound at high concentrations, but the binding to CLDN6 was stronger. Overall, 2-D11 had better binding specificity.

[0133] 4. CLDN6-2D11 cloning and humanization results

[0134] Based on the original antibody sequence information of 2D11, the homology model of the antibody was obtained by modeling and the CDRs were analyzed. A range of framework amino acids, these amino acid sites usually affect the conformation or antigen binding activity of CDR. The humanized germline was obtained through IMGT analysis. After the selected humanized germline framework was spliced ​​with the antibody CDRs, the framework region sequences of the designed humanized antibody and the original antibody were compared. By analyzing the homology modeling results of the parent antibody, amino acids similar to the surface residues of human antibodies were selected for replacement while maintaining the antibody activity and reducing heterology. The antibody humanized sequence 2D11-HM01~07 was designed, with a total of 7 sequences. The humanized antibodies designed above were gene synthesized separately and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and 3×10 CHO-S cells corresponding to CLDN3 / 4 / 6 / 9 overexpression were used. 5 The concentrations of the candidate antibodies for the CLDN6 target were 30 μg / ml, 10 μg / ml and 3 μg / ml, 100 μl / well for flow cytometry detection. The results are as follows Figure 6 As shown; Based on the above results, humanized antibodies HM3, HM4 and HM7 were selected for FACS EC50 affinity detection (3-fold gradient dilution 10 points), and the results are as follows Figure 7 As shown, the candidate antibodies all had good binding results.

[0135] 5. CLDN6 1H07 clone humanization results

[0136] Based on the original antibody sequence information of 1HM07, the homology model of the antibody was obtained by modeling and the CDRs were analyzed. A range of framework amino acids, these amino acid sites usually affect the conformation or antigen binding activity of CDR. The humanized germline was obtained by IMGT analysis. After the selected humanized germline framework was spliced ​​with the antibody CDRs, the framework region sequences of the designed humanized antibody and the original antibody were compared. By analyzing the homology modeling results of the parent antibody, amino acids similar to the surface residues of human antibodies were selected for replacement while maintaining the antibody activity and reducing heterology. The antibody humanized sequences 1H07-HM01~04 were designed, a total of 4 sequences. The humanized antibodies designed above were gene synthesized separately and subcloned into the pcDNA3.4-IgG1Fc expression vector. Control cells CHO-S and 3×10 CHO-S cells corresponding to CLDN3 / 4 / 6 / 9 overexpression were used. 5 The concentrations of the candidate antibodies for the CLDN6 target were 30 μg / ml, 10 μg / ml and 3 μg / ml, 100 μl / well for flow cytometry detection. The results are as follows Figure 8 As shown; and affinity testing was performed at 10 points with a 3-fold gradient dilution starting from 30 μg / ml. The results are shown Fig. 9 shown.

[0137] Example 2 Construction of CAR-T cells using candidate antibody sequences and verification of killing ability

[0138] 1. Construction of CAR-T cells based on cloned sequences

[0139] The corresponding candidate antibody sequence and the positive control CAR-T antibody 731B2 sequence (from US11345731 patent) were synthesized to construct the pCDH-EF1α lentiviral expression plasmid, such as Fig.10 The structure is shown in the schematic diagram. The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in the logarithmic growth phase, and the cell culture supernatant was harvested for 48-72 hours after transfection. After concentration and filtration, the CAR lentivirus was obtained and stored at -80°C for standby use. Peripheral blood mononuclear cells (PBMCs) were separated from human peripheral blood, and T cells were isolated using human CD3 / 28 magnetic beads. Viral transduction was performed within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until the 8th day. The cells were collected by centrifugation and resuspended in saline. Flow cytometry was used to identify EGFRt molecules on the surface of T cells to ensure that the positive rate was greater than 30%. If it was lower, EGFR-PE primary antibody plus PE magnetic beads were used for enrichment and sorting.

[0140] 2. Verification of CAR-T cell killing ability

[0141] 1) 2D11 and humanized 2D11-HM7, as well as control 731B2 CAR-T and negative control ctrl-T killed target CHO-S cells (overexpressing CLDN3 / 4 / 6 / 9, a total of four groups), with an effector-target ratio of 5:1. The results are as follows Fig.11 As shown, the results showed that several groups of CAR-T cells could effectively identify and kill CLDN6-CHO-S cells.

[0142] 2) Different donor negative control WT-T, 2D11 and humanized 2D11-HM4, 1H07 and humanized 1H07-HM2 CAR-T killing target cells CHO-S (overexpressing CLDN3 / 4 / 6 / 9, a total of four groups), the effector-target ratio was 1:1 and 5:1, the results are as follows Fig.12 As shown, the results showed that several groups of CAR-T cells could effectively identify and kill CLDN6-CHO-S cells.

[0143] 3) SKOV3 cell lines (adherence) overexpressing CLDN3 / 4 / 6 / 9 were constructed respectively, and RTCA was used to monitor the killing of 731B2, 2D11, 2D11HM3, and 1H07HM2 CAR-T cells, where lysis was a positive control with the addition of lysis solution, and SKOV3 was an untreated group. The specific experimental steps are as follows: 50 μL of culture medium was added to each well as a background measurement. Subsequently, the target cells were suspended in 100 μL of complete culture medium at an appropriate density (15,000 cells / well) and carefully added to each well of the E-Plate. The E-Plate was placed in the RTCA instrument, and the cell index (CI) value was continuously recorded for about 48 hours to monitor cell adhesion and growth. Among them, 12-24 hours after inoculation, pause the recording and remove the E-Plate, add 50μL T cell suspension (according to the effector-target ratio of 1:1 and 5:1) or lysis solution (positive control) to the target well, and quickly put the E-Plate back into the instrument for continued monitoring. The cell index (CI) and corresponding killing results of SKOV3 cells overexpressing CLDN3 / 4 / 6 / 9 are shown in Figure 2. Fig.13 As shown, it was demonstrated that the candidate antibody could effectively and specifically kill SKOV3 cells overexpressing CLDN6.

[0144] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An antibody specifically targeting CLDN6, characterized in that: The antibodies include: 1) HCDR1-HCDR3 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 4; or 2) HCDR1-HCDR3 of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 8; or 3) HCDR1-HCDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 12; or 4) HCDR1-HCDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16; or 5) HCDR1-HCDR3 of the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; Preferably, the numbering scheme of the CDR includes IMGT, Chothia, Kabat, AbM, Contact; Preferably, the numbering scheme of the CDRs is IMGT.

2. The antibody according to claim 1, characterized in that The sequence of HCDR1-HCDR3 of the heavy chain variable region includes: The amino acid sequences of the HCDR1-HCDR3 are shown in SEQ ID NOs: 1-3; or The amino acid sequences of the HCDR1-HCDR3 are shown in SEQ ID NOs: 5-7; or The amino acid sequences of the HCDR1-HCDR3 are shown in SEQ ID NOs: 9-11; or The amino acid sequences of HCDR1-HCDR3 are shown in SEQ ID NOs: 13-15; or The amino acid sequences of HCDR1-HCDR3 are shown in SEQ ID NOs: 17-19; Preferably, the antibody further comprises a heavy chain variable region in which part of the amino acids in the framework region of 4) or 5) are replaced with humanized amino acids; Preferably, the heavy chain variable region in which some amino acids in the framework region of 4) are replaced with humanized amino acids is as shown in any of the following groups: a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; b) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22; c) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24; Preferably, the heavy chain variable region in which some amino acids in the framework region of 5) are replaced with humanized amino acids is as shown in any of the following groups: e) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; f) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; g) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; h) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; j) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; k) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; Preferably, the antibody further comprises an Fc sequence; Preferably, the Fc sequence is as shown in SEQ ID NO: 32; Preferably, the antibody further comprises an export signal peptide; Preferably, the sequence of the export signal peptide is as shown in SEQ ID NO:

33.

3. The antibody according to claim 1, characterized in that The antibodies include single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies or bivalent antibodies; Preferably, the antibody is a single domain antibody.

4. An antibody derivative, characterized in that: The antibody derivative comprises any of the following: a. an antibody-marker conjugate, the antibody-marker conjugate comprising the antibody according to any one of claims 1 to 3 and a detectable marker conjugated thereto; b. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier; c. A chimeric antigen receptor comprising the heavy chain variable region of the antibody according to any one of claims 1 to 3.

5. The antibody derivative according to claim 4, characterized in that The detectable label comprises at least one of a radioisotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme; Preferably, the chimeric antigen receptor further comprises one or more of an extracellular hinge region, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory domain; Preferably, the extracellular hinge region is selected from the extracellular hinge regions of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the extracellular hinge region is CD8 Hinge; Preferably, the amino acid sequence of the CD8 Hinge is as shown in SEQ ID NO: 34; Preferably, the transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, 4 1BB, IgG1, IgG4, PD 1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the transmembrane domain is CD8 TM; Preferably, the amino acid sequence of the CD8 TM is as shown in SEQ ID NO: 35; Preferably, the intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and variants thereof; Preferably, the intracellular signaling domain is the CD3ζ intracellular signaling domain; Preferably, the amino acid sequence of CD3ζ is as shown in SEQ ID NO:36; Preferably, the costimulatory domain is selected from the costimulatory domains of the following molecules: 4 1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; Preferably, the costimulatory domain is 4-1BB; Preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 37; Preferably, the chimeric antigen receptor further comprises a suicide gene or a detectable tag; Preferably, the suicide gene or detectable tag comprises EGFRt; Preferably, the amino acid sequence of EGFRt is as shown in SEQ ID NO: 38; Preferably, the suicide gene or detectable tag further comprises a signal peptide of EGFRt; Preferably, the amino acid sequence of the EGFRt signal peptide is as shown in SEQ ID NO: 39; Preferably, the chimeric antigen receptor further comprises a linker; Preferably, the linker comprises a 2A peptide, an IRES; Preferably, the 2A peptides include P2A, T2A, E2A and F2A; Preferably, the linker is T2A; Preferably, the amino acid sequence of T2A is shown in SEQ ID NO:40; Preferably, the chimeric antigen receptor further comprises a signal peptide; Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

41.

6. A biomaterial comprising: (I) a nucleic acid molecule encoding the antibody according to any one of claims 1 to 3 or the antibody derivative according to claim 5; or (II) a vector comprising the nucleic acid molecule described in (I); or (III) a recombinant host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II); or (IV) A CAR-T cell comprising a chimeric antigen receptor in the antibody derivative according to any one of claims 4 to 5.

7. The biomaterial according to claim 6, characterized in that The nucleic acid molecule comprises a base sequence encoding HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO:43-58; Preferably, the base sequence encoding HCDR1-HCDR3 is shown in SEQ ID NO:59-73; Preferably, the nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 74; Preferably, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 75; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO:76; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 77; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD3ζ as shown in SEQ ID NO:78; Preferably, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO:79; Preferably, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO:80; Preferably, the nucleic acid molecule further comprises a base sequence encoding an EGFRt export signal as shown in SEQ ID NO:81; Preferably, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 82; Preferably, the nucleic acid molecule further comprises a base sequence encoding a membrane release signal as shown in SEQ ID NO:

83.

8. Use of the antibody according to any one of claims 1 to 3, the chimeric antigen receptor in the antibody derivative according to any one of claims 4 to 5, and / or the nucleic acid molecule in the biomaterial according to any one of claims 6 to 7, characterized in that: The application includes any of the following: Application in the preparation of products for detecting CLDN6 protein; Application in constructing bivalent antibodies targeting CLDN6; Application in constructing CAR-T cells that specifically target CLDN6; Application in the treatment of CLDN6-positive related diseases; Application in the preparation of products for treating CLDN6-positive related diseases.

9. The use according to claim 8, characterized in that: The products for treating CLDN6-positive related diseases include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips; Preferably, the CLDN6-positive related diseases include solid tumors; Preferably, the solid tumors include ovarian cancer, endometrial cancer, lung cancer, gastric cancer, and testicular cancer; Preferably, the ovarian cancer includes ovarian epithelial tumors, sex cord-stromal cell tumors, and germ cell tumors; Preferably, the ovarian epithelial tumor comprises an ovarian adenocarcinoma tumor; Preferably, the solid tumor is an ovarian adenocarcinoma tumor.

10. A method, comprising any of the following: (1) An in vitro method for detecting CLDN6 in a sample for non-therapeutic purposes, the method comprising: contacting the sample to be tested with the antibody according to any one of claims 1 to 3 or the antibody-marker conjugate in the composition according to claim 4, and detecting the formation of a complex between the antibody and CLDN6; (2) A method for producing the antibody according to any one of claims 1 to 3, the method comprising: culturing the recombinant host cell in the biological material according to claim 6, and isolating the antibody according to any one of claims 1 to 3 from the culture; (3) A method for promoting apoptosis of ovarian adenocarcinoma cells in vitro for non-therapeutic purposes, characterized in that the method comprises: co-culturing the CAR-T cells in the biomaterial of claim 6 with ovarian adenocarcinoma cells; Preferably, the ovarian adenocarcinoma cell is SKOV3.

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