Antibody sequences of three antibody candidates targeting human lymphocyte antigen 6 family member K (LY6K)

Abstract

The present invention relates to isolated antibodies, fragments thereof and antibody-drug conjugates that bind to Lymphocyte antigen 6 family member K (LY6K), and their use for screening for LY6K-positive malignant cells or for therapeutic targeting of LY6K-positive malignant cells.

Description

[Technical field]

[0001] The present invention relates to isolated antibodies, fragments thereof and antibody-drug conjugates that bind to Lymphocyte antigen 6 family member K (LY6K), and their use for screening for LY6K-positive malignant cells or for therapeutic targeting of LY6K-positive malignant cells. [Background technology]

[0002] Antibody-based therapies have shown tremendous clinical utility in patients with certain types of hematological and solid malignancies. However, one of the biggest challenges in antibody therapy is the need to identify target antigens that are poorly or not expressed in healthy tissues, but are specifically upregulated in cancer. Cancer / testis antigens (CTAs) are often aberrantly expressed in cancer and are absent in the majority of healthy tissues, except in testis and germline cells. Thus, CTAs are promising target antigens for immunotherapy against cancer. However, the fact that the majority of CTAs are expressed intracellularly complicates the development of antibody-based therapeutic strategies.

[0003] Thus, there is a need to provide improved antibodies. Summary of the Invention [Means for solving the problem]

[0004] To address the above challenges, we attempted to identify putative CTAs expressed on the cell surface using a bioinformatics approach. Several CTAs were identified, among which lymphocyte antigen 6 family member K (LY6K) was the most consistently upregulated target antigen in cervical cancer, tongue cancer, lung adenocarcinoma, and lung squamous cell carcinoma.

[0005] According to a first aspect, the present invention provides an isolated antibody or fragment thereof that specifically binds to at least one epitope of Lymphocyte antigen 6 family member K (LY6K), comprising: At least one light chain variable domain comprises three complementarity determining region sequences (CDR1, CDR2 and CDR3) and at least one heavy chain variable domain comprises three complementarity determining region sequences (CDR1, CDR2 and CDR3); a) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:31, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:32; or b) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 39; or c) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 45 and SEQ ID NO: 46, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 47 and SEQ ID NO: 48; An isolated antibody or fragment thereof is provided.

[0006] The determination of CDR sequences can be performed according to methods known in the art, including, but not limited to, those known as the KABAT method, the Chothia method, or the IMGT method. The set of selected CDRs may include sequences identified by more than one method, i.e., for example, some CDR sequences may be determined using the KABAT method and some CDR sequences may be determined using the IMGT method.

[0007] In some embodiments, a) the amino acid sequence of said light chain variable domain comprises six complementarity determining regions (CDRs), wherein light chain CDR1 comprises the amino acid sequence KSSQSLLASDGKTYLN as set forth in SEQ ID NO:1; light chain CDR2 comprises the amino acid sequence LVSKLDS as set forth in SEQ ID NO:2; light chain CDR3 comprises the amino acid sequence WQGSHFPLT as set forth in SEQ ID NO:3; heavy chain CDR1 comprises the amino acid sequence SYWMH as set forth in SEQ ID NO:4; heavy chain CDR2 comprises the amino acid sequence EINPSNGGANYNEKFKR as set forth in SEQ ID NO:5; heavy chain CDR3 comprises the amino acid sequence GGNFFFAY as set forth in SEQ ID NO:6; or b) the amino acid sequence of said light chain variable domain comprises six complementarity determining regions (CDRs), where light chain CDR1 comprises the amino acid sequence RSSQSLVHTDGDTYLN as set forth in SEQ ID NO: 11; light chain CDR2 comprises the amino acid sequence KVSNRFS as set forth in SEQ ID NO: 12; light chain CDR3 comprises the amino acid sequence SQNTHVYT as set forth in SEQ ID NO: 13; heavy chain CDR1 comprises the amino acid sequence SYGMS as set forth in SEQ ID NO: 14; heavy chain CDR2 comprises the amino acid sequence TISSGGTYTYYSDSVKG as set forth in SEQ ID NO: 15; heavy chain CDR3 comprises the amino acid sequence HDWDFFDY as set forth in SEQ ID NO: 16; or c) the amino acid sequence of the light chain variable domain comprises six complementarity determining regions (CDRs), whereby light chain CDR1 comprises the amino acid sequence CRSSQTILHSNGNTYLE as set forth in SEQ ID NO: 21; light chain CDR2 comprises the amino acid sequence KVSNRLS as set forth in SEQ ID NO: 22; light chain CDR3 comprises the amino acid sequence FQGSHDPFT as set forth in SEQ ID NO: 23; heavy chain CDR1 comprises the amino acid sequence NFAMN as set forth in SEQ ID NO: 24; heavy chain CDR2 comprises the amino acid sequence WINTNTGEPTYVEEFKG as set forth in SEQ ID NO: 25; and heavy chain CDR3 comprises the amino acid sequence REWRRSYYFDY as set forth in SEQ ID NO: 26.

[0008] In some embodiments, a) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:31, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:32; or b) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 39; or c) the light chain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:27, SEQ ID NO:45 and SEQ ID NO:46, and the heavy chain variable domain comprises an amino acid sequence having at least 90% sequence identity with a light chain selected from the group consisting of SEQ ID NO:28, SEQ ID NO:47 and SEQ ID NO:48.

[0009] In some embodiments, the antibodies of the invention are monoclonal murine antibodies, monoclonal humanized antibodies, or monoclonal chimeric antibodies.

[0010] The antibodies of the present invention have been found to be cytotoxic and cytotoxic in themselves. It is known that the antitumor effects of therapeutic antibodies can be exerted through downstream signaling events such as inhibiting growth and proliferation and initiating apoptosis, or by activating the patient's immune system to induce complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC) [Chalouni, C., Doll, S. J Exp Clin Cancer Res 37: 20 (2018)].

[0011] Antibody-drug conjugates (ADCs) are generally humanized or chimeric antibodies chemically linked to a cytotoxic drug, which can specifically deliver the cytotoxic drug to antigen-positive malignant cells [Chalouni, C., Doll, S. J Exp Clin Cancer Res 37: 20 (2018)]. Antibody specificity and local release of the cytotoxic drug are key parameters for enhancing antitumor activity and reducing systemic toxicity. ADCs therefore have a wider therapeutic window than traditional chemotherapy. A chemical linker is used to attach the cytotoxic drug to the antibody, and the physicochemical properties of this chemical linker determine to a large extent the pharmacokinetics of the ADC. Linkers are generally classified as “cleavable” and “non-cleavable” linkers. Linkers are designed to be highly stable in the blood circulation to limit systemic toxicity, and to be easily cleaved once the ADC reaches its intracellular destination to deliver the payload. "Cleavable" linkers are designed to release the drug by hydrolysis (reduction of disulfide bonds at low pH) or proteolysis. Linkers designed to be proteolytically degraded contain sites that are specifically recognized by certain enzymes, such as cysteine ​​proteases. "Non-cleavable" linkers rely on the degradation of the antibody itself to release the cytotoxic payload. Cytotoxic drugs are primarily classified into two types: microtubule inhibitors and DNA damaging drugs [Chalouni, C., Doll, S. J Exp Clin Cancer Res 37: 20 (2018)].

[0012] In some embodiments, the isolated antibody or fragment thereof is an antibody complex.

[0013] In some embodiments, the isolated antibody or fragment thereof is conjugated to a cytotoxic agent, a radioactive moiety, or an identifiable moiety (eg, a fluorescent tag, biotin, etc.).

[0014] According to a second aspect, the present invention provides a pharmaceutical composition comprising the isolated antibody or fragment thereof according to the first aspect or a drug conjugate thereof and a pharma- ceutically acceptable excipient, diluent, salt or carrier.

[0015] According to a third aspect, the present invention provides an isolated antibody or fragment thereof according to the first aspect, or a pharmaceutical composition according to the second aspect, for use in the treatment of LY6K-positive malignant cells.

[0016] In some embodiments, the LY6K-positive malignant cells are selected from the group including cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

[0017] According to a fourth aspect, the present invention provides the use of at least one antibody or fragment thereof according to the first aspect in the preparation of a medicament for the treatment of an LY6K-positive malignant cell disease.

[0018] In some embodiments, the LY6K-positive malignant cell disease is selected from the group consisting of cancers, such as cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

[0019] According to a fifth aspect, the present invention provides a method for treating an LY6K-positive malignant cell disease, the method comprising the step of administering to a subject an antibody or fragment thereof according to the first aspect.

[0020] In some embodiments, the LY6K-positive malignant cell disease is selected from the group consisting of cancers, such as cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

[0021] In some embodiments, the method further comprises administering an additional anti-cancer therapy selected from surgery, chemotherapy, radiation therapy, and immunotherapy.

[0022] According to a sixth aspect, the present invention provides a method for detecting LY6K-positive malignant cells in a biological sample, comprising: i) providing at least one biological sample; ii) contacting said at least one biological sample with the antibody or fragment thereof according to the first aspect; iii) detecting specific binding; and iv) comparing the specific binding of step iii) with a control. The present invention provides a method comprising:

[0023] The present invention further provides polynucleic acids encoding the light chain variable domain and the heavy chain variable domain of the antibody of the present invention.

[0024] According to an eighth aspect, the present invention provides an isolated nucleic acid molecule comprising: (a) encoding at least one light chain variable domain of the antibody of claim 2 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 31, and (b) encoding at least one heavy chain variable domain of the antibody of claim 1 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 32; or (c) encoding at least one light chain variable domain of the antibody of claim 2 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; and (d) encoding at least one heavy chain variable domain of the antibody of claim 1 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 39; or (e) encoding at least one light chain variable domain of the antibody of claim 2 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 45 and SEQ ID NO: 46; and (f) encoding at least one heavy chain variable domain of the antibody of claim 1 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 47 and SEQ ID NO: 48. Isolated nucleic acid molecules are provided.

[0025] In some embodiments, i) at least one nucleic acid sequence according to (a) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:9 and / or SEQ ID NO:33; and at least one nucleic acid sequence according to (b) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:10 and / or SEQ ID NO:34; or ii) the nucleic acid sequence according to (c) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:19, SEQ ID NO:40, SEQ ID NO:41 and / or SEQ ID NO:42; and at least one nucleic acid sequence according to (d) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:20, SEQ ID NO:43 and / or SEQ ID NO:44; or iii) the nucleic acid sequence described in (e) has at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:29, SEQ ID NO:49 and / or SEQ ID NO:50; and at least one nucleic acid sequence described in (f) has at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO:30, SEQ ID NO:51 and / or SEQ ID NO:52.

[0026] According to a ninth aspect, the present invention provides an expression vector comprising at least one isolated nucleic acid molecule according to the eighth aspect.

[0027] According to a tenth aspect, the present invention provides a host cell comprising an expression vector according to the ninth aspect.

[0028] According to an eleventh aspect, the present invention provides a kit comprising at least one antibody or fragment thereof according to the first aspect.

[0029] The present invention is not limited to the specific embodiments detailed below. [Brief description of the drawings]

[0030] [Figure 1A-C] Cell surface expression of LY6K is shown. (A) LY6K is expressed on the cell surface of human cancer cell lines. Cell surface expression of LY6K was assessed by flow cytometry. Representative histograms of two breast cancer cell lines (MDA-MB-231 and MCF7), one lung cancer cell line (Calu-1), and one liver cancer cell line (HUH7) are shown. LY6K (black solid line) was detected on the cell surface of breast and lung cancer cells, but not liver cells. Isotype antibodies were used as controls (filled histograms). (B) Quantification of the expression of LY6K on the cell surface of human cancer cell lines. High expression of LY6K was detected on the cell surface of cervical, head and neck, and lung cancer cell lines, as well as on the cell surface of some breast cancer cell lines. Low expression was found in hematological, colon, liver, and pancreatic cancer cell lines. Tissues of origin of cancer cell lines are shown. Data show the mean ± sem of three independent experiments. (C) Detection of LY6K on the cell surface by confocal microscopy. The cell membranes of MDA-MB-231 and MCF7 cells (both breast), Calu-1 cells (lung), and HUH7 cells (liver) were stained with a red cell membrane dye (CellMask) and green anti-LY6K antibody. Scale bar indicates 20 μm.

[0031] [Diagram 2] We show that the expressed monoclonal anti-LY6K antibody binds to recombinant LY6K. The heavy and light chains from the hybridomas were cloned into pTT5LnX-MoG1v2 and pTT5LnX-Kappa expression vectors, respectively. The expressed antibody was checked for purity by running on an SDS-PAGE gel and tested in an ELISA binding assay with recombinant LY6K protein. Recombinant LY6K had a histidine tag and a Strep tag, so His and Strep were included as controls to ensure that the antibody did not bind to these tags.

[0032] [Diagram 3] Figure 1 shows the evaluation of real-time binding properties of antibodies to recombinant LY6K. Representative sensorgrams showing the binding of various concentrations of antibodies to immobilized recombinant LY6K are shown. The equilibrium dissociation constants (KD) indicate that all antibody candidates have high binding affinity to LY6K at low nanomolar levels. The highest number of binding sites (Bmax) was observed for the 28C1 antibody, followed by the G57 antibody, and then the 22C-G8 antibody. Standard errors are shown in brackets.

[0033] [Figure 4] Anti-LY6K antibodies bind to various human cancer cell lines. Flow cytometry analysis was performed to evaluate the binding of antibody candidates to various human cancer cell lines derived from different tissues. (A): Breast cancer; (B): Cervical cancer; (C): Lung cancer; (D): Pancreatic cancer. The percentage of LY6K expressing cells compared to isotype control is shown and each cell line is indicated.

[0034] [Diagram 5] Anti-LY6K antibodies 22C-G8 and 28C1 show higher binding to LY6K expressed on the surface of Ca Ski cells than four commercially available antibodies. Binding to LY6K was assessed by flow cytometry on the cervical cancer cell line Ca Ski, and representative histograms are shown. Isotype antibodies (histogram on the left) were used as controls for staining with LY6K antibodies (histogram on the right). 22C-G8 and 28C1 showed the highest binding to LY6K, 90.4% and 95.6%, respectively. The next highest binding was clone G-11 (Santa Cruz) at 85.8%, followed by clone #750747 (R&D Systems) at 27.7%, then clone CL2435 (Sigma-Aldrich) at 4.64%, and then clone CL2433 (Invitrogen) at 0.49%.

[0035] [Figure 6] Antibody-mediated complement-dependent cytotoxicity of cervical cancer cells. Antibody candidates G57 and 28C1 induced complement-dependent cytotoxicity (CDC) against cervical cancer cells (Ca-Ski) at concentrations of 10 μg / ml and 50 μg / ml. In contrast, only slight CDC killing was observed with the antibody candidate 22C-G8 at a concentration of 50 μg / ml. Isotype control (ITC) was used as a negative control.

[0036] [Figure 7] Immunohistochemistry analysis of tongue and lung tissue microarrays using anti-LY6K antibody. Formalin-fixed, paraffin-embedded tongue squamous cell carcinoma (SCC), lung adenocarcinoma, and lung SCC were stained with antibody candidate 28C1, whose binding was detected with an HRP-conjugated secondary antibody (dark grey staining area). Samples were counterstained with hematoxylin (grey staining area).

[0037] [Figure 8] Figure 1 shows humanization of anti-LY6K antibodies. Humanized forms of anti-LY6K antibodies 22C-G8, G57 and 28C1 were expressed and run on SDS-PAGE gel to confirm their purity. ELISA binding assay confirmed the binding ability of humanized antibodies to recombinant LY6K, and flow cytometry assay confirmed the binding ability of humanized antibodies to cancer cell line Calu-1.

[0038] [Figure 9] Binding profiles of humanized 22C-G8 and humanized 28C1 antibodies against a panel of cancer cell lines are shown. Humanized 22C-G8 (dark grey solid line) and humanized 28C1 (grey solid line) antibodies retained binding to LY6K expressed on the surface of human tumor cell lines derived from various organs including breast, cervix, lung and head and neck. LY6K cell surface expression was assessed by flow cytometry staining with Hu22CG8 or Hu28C1 antibodies and detected using an anti-human AlexaFluor 647 secondary antibody.

[0039] [Figure 10] Figure 1 shows the internalization of humanized 22C-G8 antibody into Ca Ski cells. (A) Confocal microscopy showed the presence of red fluorescence in Ca Ski cells, indicating that humanized 22C-G8 antibody is internalized. Isotype or humanized 22C-G8 antibody was covalently labeled with pHRodo Deep Red dye to form a complex that fluoresces only in acidic late endosomes and lysosomes. Labeled antibody (10 μg / mL) was incubated with Ca Ski cells for 18 hours, stained with green cell membrane dye (CellMask) and Hoechst 33342, and imaged with an FV3000 confocal microscope. Scale bar indicates 40 μm. (B) Concentration- and time-dependent increase in antibody internalization in Ca Ski cells. Humanized 22CG8 antibody and isotype control were labeled with Incucyte® Human Fabfluor-pH Red Antibody Labeling Reagent and added to Ca Ski cells at various concentrations. High definition (HD) phase images and red fluorescent images were taken at 20x magnification every hour up to 50 hours. Data were normalized to t=0 and isotype control. Data are shown as the mean ± standard deviation of three replicates.

[0040] [Figure 11]Humanized 22C-G8 and humanized 28C1 antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). (A) Increased killing of HEK293T-Ly6K-overexpressing cells in the presence of Hu22CG8 and Hu28C1 antibodies at three E:T ratios compared to isotype control. HEK293T-Ly6K-overexpressing cells were seeded on E-plates and cultured overnight, and each antibody at a concentration of 25 μg / mL was added to the cells and incubated. NK effector cells isolated from PBMCs by immunomagnetic negative selection were then added to each well at three E:T ratios of 4:1, 8:1, or 16:1. Cell killing was monitored for 60 hours using the RTCA xCELLigence system. (B) Specific cytotoxicity induced by Hu22CG8, Hu28C1, or the combination of Hu22CG8 and Hu28C1 antibodies. Specific cytotoxicity was calculated by normalizing each cytotoxicity curve to an isotype control curve and plotted over time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] For easy reference, a list of the references cited herein is provided after the Examples, all of which are incorporated herein by reference in their entirety.

[0042] Definition of Terms For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0043] As used herein, the terms "comprising" or "including" are to be construed as specifying the presence of the features, integers, steps, or ingredients described herein denoted by these terms, but not excluding the presence or addition of one or more other features, integers, steps, or ingredients, or groups thereof. In the context of this disclosure, the terms "comprising" or "including" also encompass the meaning "consisting of." Thus, variations of the term "comprising," such as "comprise" or "comprises," and variations of the term "including," such as "include" or "includes," have similarly various meanings.

[0044] As used herein, "antibody" refers to an immunoglobulin or intact molecule that binds to a specific epitope, and fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, single chain variable region fragments (scFv), and Fab fragments, Fab' fragments, F(ab)' fragments, and / or F(v) portions of full-length antibodies. "Monoclonal antibodies" may also be referred to as "Mab". The antibodies of the present invention include 22C-G8, G57, and 28C1 antibodies produced by hybridoma cell lines or recombinant technology. 22C-G8, G57, and 28C1 antibodies may be monoclonal, polyclonal, or single chain antibodies, or fragments thereof that retain the antigen-binding function of the parent antibody. The 22C-G8, G57 and 28C1 antibodies specifically bind to LY6K and include monoclonal, polyclonal and single chain antibodies, as well as fragments thereof which retain the antigen-binding function of the parent antibody.

[0045] As used herein, "antibody fragment" refers to an incomplete or isolated portion derived from the full-length sequence of an antibody and retaining the antigen-binding function of the parent antibody. Antibody fragments include scFv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Fragments of 22C-G8, G57 and 28C1 antibodies are encompassed by the present invention as long as they retain the desired affinity of the full-length antibody.

[0046] As used herein, a "humanized antibody" refers to at least one antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered to increase similarity to a human antibody, but which retains the original binding ability. Such antibodies are also shown in Table 1.

[0047] As used herein, "hybridoma" refers to a cell that is created to mass-produce a desired antibody. For example, to create at least one hybridoma, B cells are taken from the spleen of an animal challenged with a relevant antigen and fused with at least one immortalized cell. This fusion is achieved by increasing the permeability of the cell membrane. The fused hybrid cell (called a "hybridoma") grows rapidly and indefinitely to produce at least one antibody.

[0048] As used herein, "isolated" means that a biological component (e.g., a nucleic acid, a peptide, or a protein) is substantially separated from, produced substantially separately from, or substantially purified from other biological components (i.e., other chromosomal DNA, extrachromosomal DNA, chromosomal RNA, extrachromosomal RNA, and proteins) in the cells of an organism that naturally produces the biological component. Thus, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. Furthermore, the term "isolated" includes nucleic acids, peptides, and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0049] In this specification, the term "sample" is used in the broadest sense. Biological samples suspected to contain LY6K may include body fluids; cell extracts; chromosomes, organelles or cell membranes isolated from cells; cells; genomic DNA, RNA or cDNA (in solution or bound to solid support); tissues; tissue prints, etc. More specifically, samples of interest are those that may contain LY6K-positive malignant cells.

[0050] In this specification, "subject" is defined as a vertebrate, specifically refers to a mammal, more specifically refers to a human. More specifically, for research purposes, the subject may be at least one animal model, such as mouse or rat. Specifically, the subject to be treated for LY6K-related disease may be a human having LY6K-positive cancer cells.

[0051] The compositions or combinations of the present invention are usually administered as pharmaceutical preparations in admixture with pharma- ceutically acceptable adjuvants, diluents or carriers, which may be selected with due consideration of the intended route of administration and standard pharmaceutical practice. Such pharma- ceutical acceptable carriers may be chemically inert to the active compounds and may have no adverse side effects or toxicity under the conditions of use. Suitable pharmaceutical preparations may be, for example, those described in Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, parenterally acceptable aqueous solutions may be used, which are pyrogen-free and have the required pH, isotonicity and stability. Suitable aqueous solutions are well known to those skilled in the art, and numerous methods are described in various literature. A brief description of drug delivery methods is also described, for example, in Langer (Science 249: 1527 (1990)).

[0052] Alternatively, the preparation of suitable formulations may be routinely carried out by one skilled in the art using conventional techniques and / or in accordance with standard and / or generally accepted pharmaceutical practice.

[0053] The amount of the composition or combination contained in the pharmaceutical preparation used according to the present invention depends on various factors, such as the severity of the pathology being treated, the specific patient being treated, and the compound or compounds being used. In some embodiments, BMC-VLPs present antigen molecules on their surface and function as vaccines. In any case, the amount of the composition or combination contained in the preparation can be routinely determined by those skilled in the art.

[0054] For example, oral solid compositions such as tablets and capsules may contain 1-99% (w / w) active ingredient; 0-99% (w / w) diluent or filler; 0-20% (w / w) disintegrant; 0-5% (w / w) lubricant; 0-5% (w / w) glidant; 0-50% (w / w) granulating or binding agent; 0-5% (w / w) antioxidant; and 0-5% (w / w) dye. Controlled release tablets may further contain 0-90% (w / w) release-controlling polymer.

[0055] Parenteral preparations (solutions or suspensions for injection, or solutions for infusion) may contain 1-50% (w / w) of the active ingredient; 50% (w / w)-99% (w / w) of a liquid or semisolid carrier or solvent (e.g., a solvent such as water); and 0-20% (w / w) of one or more other additives (e.g., buffers, antioxidants, suspension stabilizers, isotonicity agents, preservatives, etc.).

[0056] However, in the context of the present invention, the dose administered to a mammal, particularly the dose administered to a human, should be sufficient to exert a therapeutic response in the mammal within a reasonable time frame.Those skilled in the art will recognize that the selection of the exact dose and composition and optimal delivery regimen is influenced by the pharmacological properties of the formulation, the characteristics and severity of the pathology to be treated, the physical condition and mental function of the recipient, the potency of the specific compound, the age, condition, weight, sex and response of the patient to be treated, etc.

[0057] Having generally described the invention, the same may be more readily understood by reference to the following examples which are given for purposes of illustration and are not intended to be limiting. EXAMPLES

[0058] Standard molecular biology techniques known in the art that are not specifically described herein were generally as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

[0059] Example 1: A bioinformatics approach was used to identify cancer / testis antigens (CTAs) that are putatively expressed on the cell surface. Several CTAs that are putatively expressed on the cell membrane were identified, among which LY6K was the most consistently upregulated target antigen in cervical cancer, tongue cancer, lung adenocarcinoma, and lung squamous cell carcinoma. LY6K is a glycophosphatidylinositol (GPI)-anchored protein, and is therefore likely to be expressed on the cell surface. Flow cytometry analysis and confocal microscopy were used to evaluate the presence or absence of LY6K on the cell membrane of various human cancer cell lines (Figure 1). The results showed that LY6K was present on the cell surface of cervical cancer cell lines, head and neck cancer cell lines, and lung cancer cell lines, as well as some breast cancer cell lines tested (Figures 1A and 1B). LY6K was not expressed or was expressed at very low levels in hematological, colon, liver, and pancreatic cancer cell lines. To confirm these findings, two breast cancer cell lines (MDA-MB-231 and MCF7), one lung cancer cell line (Calu-1), and one liver cancer cell line (HUH7) were stained with an erythrocyte membrane dye (CellMask) and anti-LY6K antibody and observed by confocal microscopy (Fig. 1C). The LY6K signal was clearly shown to overlap with the cell membrane staining, confirming the previous findings that LY6K is localized on the cell membrane.

[0060] Example 2: Next, three unique antibodies targeting LY6K (referred to as 22C-G8, G57, and 28C1, respectively) were constructed using hybridoma technology. The antibody variable fragments of the heavy chain and the antibody variable fragments of the light chain were cloned into expression vectors. The purity of the expressed antibodies was confirmed by running them on an SDS-PAGE gel (Figure 2), and their binding ability to recombinant LY6K was tested by an ELISA binding assay (Figure 2). Next, the binding affinity to the immobilized LY6K antigen was evaluated using a quartz crystal microbalance (QCM) biosensor. The sensorgrams of each antibody are shown in Figure 3, along with their respective equilibrium dissociation constants (KD). The KD of all antibodies was in the low nanomolar range, indicating that all of these antibodies bind strongly to LY6K. The maximum number of binding sites (B max ) was the most abundant antibody candidate, suggesting that this antibody binds to a highly abundant or more easily accessible epitope.

[0061] Example 3: Next, we tested the binding ability of the 22C-G8, G57 and 28C1 antibodies to human tumor cell lines derived from breast, cervix, lung and pancreas. The 22C-G8 antibody showed high binding to all cell lines tested, and the G57 antibody also bound to all cell lines tested, but the binding of the G57 antibody to each cell line was lower than that of the 22C-G8 antibody. The 28C1 antibody bound strongly only to the MDA-MB-157 cell line (Figure 4A). Both antibodies also bound strongly to all cervical cancer cell lines tested (Figure 4B). Binding to lung cancer cell lines was variable. The 22C-G8 antibody bound strongly to all cell lines tested, while the 28C1 antibody showed a very weak binding profile. The strongest binding was observed to Calu-1 cells. The G57 antibody also bound strongly to Calu-1 cells but only weakly to A549 and H2170 cells (Figure 4C). Pancreatic cell lines were used as negative controls because they do not express sufficient amounts of LY6K on their cell surface. Except for the 22CG8 antibody, which bound strongly to the CRL2553 cell line, the antibody candidates hardly bound to any of the three pancreatic cell lines tested (Figure 4D).

[0062] Example 4: The binding of 22C-G8 and 28C1 antibodies to LY6K expressed on the cervical cancer cell line Ca Ski was compared with commercially available LY6K antibody clones. Flow cytometry analysis showed that both 22C-G8 and 28C1 antibodies showed superior binding to LY6K expressed on the cell surface of Ca Ski cells compared with commercially available antibodies (Figure 5). 22C-G8 and 28C1 antibodies were among the antibodies that showed the highest binding to LY6K. Clone G-11 (Santa Cruz) showed similar binding to LY6K as these antibodies. In comparison with these antibodies, the other antibodies, i.e., clone #750747 (R&D Systems), clone CL2435 (Sigma-Aldrich), and clone CL2433 (Invitrogen), showed very weak binding profiles. This result was significant because strong binding to LY6K expressed in its native conformation on the cell surface of cancer cells is a key prerequisite for therapeutic application of the antibody.

[0063] Example 5: We further evaluated whether anti-LY6K antibodies could kill tumor cells through complement-dependent cytotoxicity. G57 and 28C1 antibodies were found to be able to kill cervical cancer cells (Ca-Ski cells) at antibody concentrations of 10 μg / ml and 50 μg / ml. A slight killing effect was observed with 22C-G8 antibody at a concentration of 50 μg / ml (Figure 6).

[0064] Example 6: To use anti-LY6K antibodies in cancer diagnosis (and its treatment), antibody binding to healthy tissues must be minimized. With this in mind, we performed immunohistochemical analysis of tongue squamous cell carcinoma (SCC), lung adenocarcinoma, and lung SCC tissue sections, as well as healthy tongue and lung tissue sections (Figure 7). The candidate anti-LY6K antibody, 28C1, stained malignant tongue and lung tissues, but not these healthy tissues, indicating that the 28C1 antibody can distinguish between malignant and healthy tissue samples.

[0065] Example 7: Each mouse antibody was humanized by grafting the complementarity determining regions (CDRs) of the above mouse antibodies into human framework regions. These humanized antibodies were expressed and their purity was confirmed by SDS-PAGE (Figure 8). The binding ability of each humanized antibody to recombinant LY6K was evaluated by ELISA assay. Humanized 22C-G8 and humanized 28C1 antibodies showed similar binding ability to recombinant LY6K as their mouse antibody forms, whereas humanized G57 antibody showed reduced binding affinity to LY6K. Flow cytometry analysis showed that humanized 22C-G8 antibody retained strong binding ability to Calu-1 cells, whereas humanized forms of G57 and 28C1 antibodies lost some of their affinity.

[0066] We further verified the binding ability of the humanized antibodies to LY6K expressed on the surface of cancer cells and confirmed that the humanized 22C-G8 and humanized 28C1 antibodies retained binding to LY6K expressed on the surface of a panel of human tumor cell lines derived from various organs, including breast, cervix, lung and head and neck (Figure 9).

[0067] Example 8: To further explore the potential of the humanized anti-LY6K antibody as an antibody-drug conjugate, we tested whether the antibody could be internalized into cells. The fluorescent signal was distributed in a punctate manner in the microscopic images, indicating that the 22C-G8 antibody was localized in endosomes, which indicated that the 22C-G8 antibody was internalized into Ca Ski cells (Figure 10A). Furthermore, real-time monitoring of the fluorescence intensity over time showed that the internalization of the antibody was concentration- and time-dependent (Figure 10B). Since the internalization of the antibody is a necessary condition for the incorporation of the antibody into an antibody-drug conjugate, this indicates that the anti-LY6K antibody has a high potential for use as an antibody-drug conjugate for the delivery of cytotoxic drugs to LY6K-positive malignant cells.

[0068] Because antibody-dependent cellular cytotoxicity (ADCC) is a crucial mechanism underlying immunotherapeutic approaches using targeted antibodies, we examined the Hu22CG8 and Hu28C1 antibodies for their potential ability to target and kill LY6K-expressing cells via ADCC (Figure 11). The results showed that both humanized antibodies mediated ADCC and effectively eliminated tumor cells in cells highly expressing LY6K, with the Hu28C1 antibody showing higher cytotoxicity. This finding was significant because it suggested that LY6K expressed on tumor cells could be targeted by monoclonal antibodies in the presence of effector immune cells.

[0069] [Table 1] TIFF2025502247000003.tif228155TIFF2025502247000004.tif232155TIFF20255022470 00005.tif232155TIFF2025502247000006.tif227156TIFF2025502247000007.tif182156

[0070] summary We found that LY6K is a CTA expressed on the cell surface of various human cancers, making it an attractive target antigen for antibody-based methods, and we generated three unique anti-LY6K antibodies and provided evidence that these antibodies may be useful in cancer diagnosis and / or therapy.

[0071] References The listing of previously published documents cited in this specification and the discussion therein are not necessarily acknowledged to form part of the state of the art or common general knowledge. 1. Chalouni, C., Doll, S. Fate of Antibody-Drug Conjugates in Cancer Cells. J Exp Clin Cancer Res 37, 20 (2018). 2. Langer R. New methods of drug delivery. Science. 249(4976): 1527-33 (1990). 3. Remington J.P. and Gennaro A.R. The Science and Practice of Pharmacy, 19 th ed., Easton, PA : Mack Publishing (1995). 4. Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

Claims

1. 1. An isolated antibody or fragment thereof that specifically binds to at least one epitope of Lymphocyte antigen 6 family member K (LY6K), at least one light chain variable domain and at least one heavy chain variable domain, a) the amino acid sequence of the light chain variable domain comprises six complementarity determining regions (CDRs), where light chain CDR1 comprises the amino acid sequence KSSQSLLASDGKTYLN as set forth in SEQ ID NO:1; light chain CDR2 comprises the amino acid sequence LVSKLDS as set forth in SEQ ID NO:2; light chain CDR3 comprises the amino acid sequence WQGSHFPLT as set forth in SEQ ID NO:3; heavy chain CDR1 comprises the amino acid sequence SYWMH as set forth in SEQ ID NO:4; heavy chain CDR2 comprises the amino acid sequence EINPSNGGANYNEKFKR as set forth in SEQ ID NO:5; heavy chain CDR3 comprises the amino acid sequence GGNFFFAY as set forth in SEQ ID NO:6; or b) the amino acid sequence of said light chain variable domain comprises six complementarity determining regions (CDRs), whereby light chain CDR1 comprises the amino acid sequence RSSQSLVHTDGDTYLN as set forth in SEQ ID NO: 11; light chain CDR2 comprises the amino acid sequence KVSNRFS as set forth in SEQ ID NO: 12; light chain CDR3 comprises the amino acid sequence SQNTHVYT as set forth in SEQ ID NO: 13; heavy chain CDR1 comprises the amino acid sequence SYGMS as set forth in SEQ ID NO: 14; heavy chain CDR2 comprises the amino acid sequence TISSGGTYTYYSDSVKG as set forth in SEQ ID NO: 15; and heavy chain CDR3 comprises the amino acid sequence HDWDFFDY as set forth in SEQ ID NO: 16; or c) the amino acid sequence of the light chain variable domain comprises six complementarity determining regions (CDRs), where light chain CDR1 comprises the amino acid sequence CRSSQTILHSNGNTYLE as set forth in SEQ ID NO: 21; light chain CDR2 comprises the amino acid sequence KVSNRLS as set forth in SEQ ID NO: 22; light chain CDR3 comprises the amino acid sequence FQGSHDPFT as set forth in SEQ ID NO: 23; heavy chain CDR1 comprises the amino acid sequence NFAMN as set forth in SEQ ID NO: 24; heavy chain CDR2 comprises the amino acid sequence WINTNTGEPTYVEEFKG as set forth in SEQ ID NO: 25; and heavy chain CDR3 comprises the amino acid sequence REWRRSYYFDY as set forth in SEQ ID NO: 26; An isolated antibody or fragment thereof.

2. a) the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:31, and the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:32; b) the light chain comprises an amino acid sequence selected from the group comprising SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the heavy chain variable domain comprises an amino acid sequence selected from the group comprising SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 39; or c) the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 45 and SEQ ID NO: 46, and the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 47 and SEQ ID NO: 48; 2. An isolated antibody or fragment thereof according to claim 1.

3. 3. The isolated antibody or fragment thereof of claim 1 or 2, which is an antibody complex.

4. The isolated antibody or fragment thereof of claim 3 , which is conjugated to a cytotoxic drug, a radioactive moiety or an identifiable moiety.

5. The isolated antibody or fragment thereof according to any one of claims 1 to 4, wherein the antibody is a monoclonal murine antibody, a monoclonal humanized antibody, or a monoclonal chimeric antibody.

6. A pharmaceutical composition comprising the isolated antibody or fragment thereof of any one of claims 1 to 5 and a pharma- ceutically acceptable excipient, diluent, salt or carrier.

7. The isolated antibody or fragment thereof according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 6, for use in the treatment of LY6K-positive malignant cells.

8. The isolated antibody, fragment thereof or pharmaceutical composition of claim 7, wherein the LY6K-positive malignant cells are selected from the group including cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

9. Use of at least one antibody or fragment thereof according to any one of claims 1 to 5 in the preparation of a medicinal product for the treatment of an LY6K-positive malignant cell disease.

10. The use according to claim 9, wherein the LY6K-positive malignant cell disease is selected from the group consisting of cancers such as cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

11. A method for treating an LY6K-positive malignant cell disease, comprising the step of administering to a subject an antibody or a fragment thereof described in any one of claims 1 to 5.

12. The method of claim 11, wherein the LY6K-positive malignant cell disease is selected from the group consisting of cancers such as cervical cancer, lung adenocarcinoma, lung squamous cell carcinoma (SCC), tongue squamous cell carcinoma (SCC), head and neck cancer, and breast cancer.

13. 1. A method for detecting LY6K-positive malignant cells in a biological sample, comprising: i) providing at least one biological sample; ii) contacting said at least one biological sample with an antibody or fragment thereof according to any one of claims 1 to 3 and 5; iii) detecting specific binding; and iv) comparing the specific binding of step iii) with a control. A method comprising:

14. 1. An isolated nucleic acid molecule comprising: (a) at least one light chain variable domain of the antibody of claim 2 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 31; and (b) at least one heavy chain variable domain of the antibody of claim 1 or 5, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 32; and or (c) at least one light chain variable domain of the antibody of claim 2 or 5 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; and (d) at least one heavy chain variable domain of the antibody of claim 1 or 5, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 38, and SEQ ID NO: 39; and or (e) at least one light chain variable domain of the antibody of claim 2 or 5, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 45, and SEQ ID NO: 46; and (f) at least one heavy chain variable domain of the antibody of claim 1 or 5, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 47, and SEQ ID NO: 48; and To code An isolated nucleic acid molecule.

15. i) at least one nucleic acid sequence according to (a) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:9 and / or SEQ ID NO:33; and at least one nucleic acid sequence according to (b) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:10 and / or SEQ ID NO:34; or ii) the nucleic acid sequence according to (c) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:19, SEQ ID NO:40, SEQ ID NO:41 and / or SEQ ID NO:42; and at least one nucleic acid sequence according to (d) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:20, SEQ ID NO:43 and / or SEQ ID NO:44; or iii) the nucleic acid sequence according to (e) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:29, SEQ ID NO:49 and / or SEQ ID NO:50; and at least one nucleic acid sequence according to (f) has at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:30, SEQ ID NO:51 and / or SEQ ID NO:52; 15. The isolated nucleic acid molecule of claim 14.

16. 16. An expression vector comprising at least one isolated nucleic acid molecule according to claim 14 or 15.

17. A host cell comprising the expression vector of claim 16.

18. A kit comprising at least one antibody or fragment thereof according to any one of claims 1 to 5.

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