Anti-ly6g6d antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The application belongs to the technical field of biology, and provides an anti-LY6G6D antibody or antigen binding fragment thereof and application thereof. The antibody or antigen binding fragment thereof provided by the application has high affinity with human LY6G6D, and has excellent drug development prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to anti-LY6G6D antibodies or their antigen-binding fragments and their uses. Background Technology

[0002] Colorectal cancer (CRC) ranks second in cancer mortality worldwide. However, current chemotherapy drugs for CRC face clinical challenges such as low efficacy, significant side effects, and resistance reversal, leading to poor prognosis for CRC patients. Existing targeted therapies are primarily immune checkpoint inhibitors (ICIs), which have shown clinical efficacy against a subset of colorectal cancer (dMMR or MSI-H). However, stage 4 dMMR or MSI-H tumors account for only about 2% to 4% of all mCRC, and the vast majority of patients do not benefit from ICI treatment.

[0003] Lymphocyte antigen 6 complex locus G6D (LY6G6D), also known as LY6-D, LY6-G, G6D, NG25, MEGT1, and C6orf23, is a cluster of leukocyte antigens located in the MHC class III region of chromosome 6. It is a phosphatidylinositol-anchored cell surface protein encoding a 133-amino acid residue protein with a molecular weight of approximately 13.7 kDa. It attaches to the cell membrane via glycosylphosphatidylinositol (GPI). LY6G6D is a dual-mechanism target, exhibiting TSA-specific expression in CRC cells, with minimal expression in normal tissues; simultaneously, it can regulate the immune JAK / STAT axis. JAK / STAT inhibitors can increase CRC cell death by targeting the STAT5 / LY6G6D axis.

[0004] Compared with normal colorectal tissue, LY6G6D is highly expressed in both primary and metastatic colorectal tumors. High LY6G6D expression is associated with high infiltration of immunosuppressive cells; in colon cancer with reduced CD8+ T lymphocytes, LY6G6D expression and STAT5 activation are enhanced. STAT5 knockout significantly reduces LY6G6D levels, indicating that STAT5 regulates LY6G6D expression. Furthermore, the expansion of LY6G6D-positive MSS cells (which can be transmitted through myeloid-derived suppressor cells, MDSCs) inhibits T cell proliferation. Similarly, in CRC, inhibitors of p38α MAPK and knockdown of the DNA methyltransferase DNMT1 lead to decreased LY6G6D expression. In metastatic CRC, LY6G6D hypermethylation predicts resistance to first-line FOLFOX (the drugs leucovorin, fluorouracil (5-FU), and oxaliplatin). Therefore, differentially high expression of LY6G6D in colorectal cancer is closely related to cancer development and is expected to serve as a potential predictive indicator or therapeutic target for colorectal cancer. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an isolated antibody or its antigen-binding fragment that binds to the human lymphocyte antigen 6 complex locus G6D (LY6G6D).

[0006] In a first aspect, the present invention provides an anti-LY6G6D antibody or an antigen-binding fragment thereof having binding specificity to human lymphocyte antigen 6 complex site G6D (LY6G6D), said antibody or antigen-binding fragment thereof comprising:

[0007] The three CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 25 and the three CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 29; or

[0008] The three CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 1, and the three CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 5; or

[0009] The three CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 9, and the three CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 13; or

[0010] The three CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 17 and the three CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 21.

[0011] Those skilled in the art can determine the CDR sequence based on the illustrated heavy chain and light chain variable region sequences using methods known in the art. These known methods may include the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme. It is well known in the art that CDR sequences obtained using different schemes for the same variable region sequence will differ. Furthermore, those skilled in the art will understand that CDR regions obtained using different schemes are all within the scope of protection of this invention.

[0012] In some implementations, the Kabat scheme is used to determine the CDR region sequence.

[0013] In some embodiments, the present invention provides an anti-LY6G6D antibody or antigen-binding fragment thereof having binding specificity to human lymphocyte antigen 6 complex site G6D (LY6G6D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein...

[0014] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 26.

[0015] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 27.

[0016] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 28.

[0017] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 30.

[0018] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 31.

[0019] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 32; or

[0020] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2.

[0021] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 3.

[0022] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 4.

[0023] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 6.

[0024] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 7.

[0025] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 8; or

[0026] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 10.

[0027] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 11.

[0028] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 12.

[0029] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 14.

[0030] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 15.

[0031] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or

[0032] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 18.

[0033] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 19.

[0034] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 20.

[0035] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 22.

[0036] The amino acid sequence of LCDR2 is shown in SEQ ID NO: 23.

[0037] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 24.

[0038] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 25, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 25, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 29, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 29, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or

[0039] The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 1, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 5, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or

[0040] The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 9, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 9, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 13, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 13, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or

[0041] The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 17, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 21, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 21, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0042] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0043] In some implementations, the light chain constant region is a κ chain or λ chain constant region.

[0044] In some implementations, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE, or IgD class.

[0045] In some implementations, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclasses.

[0046] In some embodiments, the antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

[0047] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region being shown in SEQ ID NO: 33, and the amino acid sequence of the light chain constant region being shown in SEQ ID NO: 34.

[0048] In some implementations, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody.

[0049] In some implementations, the antigen-binding fragment is F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv.

[0050] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibodies), a polyclonal antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0051] In some embodiments, the antibody is a chimeric antibody, and the antibody or its antigen-binding fragment comprises a light chain and a heavy chain.

[0052] In some embodiments, the antibody or its antigen-binding fragment is a linear antibody or its antigen-binding fragment, including the aforementioned heavy chain variable region and light chain variable region, which are linked by a linker peptide.

[0053] In some embodiments, the linker peptide can be any linker peptide known in the art for use with scFv. In some preferred embodiments, the linker peptide is 3×G4S, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0054] In some embodiments, the linear antibody or its antigen-binding fragment further includes a CD8α signal peptide, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain.

[0055] In some embodiments, from the N-terminus to the C-terminus, the linear antibody or its antigen-binding fragment comprises, in sequence, a CD8α signal peptide, a heavy chain variable region, a linker peptide, a light chain variable region, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain.

[0056] In some embodiments, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, 1, 9 or 17, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 43, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29, 5, 13 or 21, the amino acid sequence of the CD8α hinge and CD8α transmembrane region is shown in SEQ ID NO: 37, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 39, and the amino acid sequence of the CD3 partial intracellular domain is shown in SEQ ID NO: 41.

[0057] In some embodiments, the linear antibody or its antigen-binding fragment comprises, from the N-terminus to the C-terminus, a sequentially linked CD8α signal peptide, a heavy chain variable region, a linker peptide, a light chain variable region, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain, wherein the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 43, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29, the amino acid sequences of the CD8α hinge and the CD8α transmembrane region are shown in SEQ ID NO: 37, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 39, and the amino acid sequence of the partial CD3 intracellular domain is shown in SEQ ID NO: 41.

[0058] In a second aspect, the present invention provides a biomaterial selected from (a) to (c) below.

[0059] (a) A nucleic acid molecule that encodes any of the antibodies or antigen-binding fragments described above.

[0060] In some embodiments, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0061] In some implementations, the nucleic acid molecule is an isolated nucleic acid molecule.

[0062] (b) A recombinant vector containing the aforementioned nucleic acid molecules.

[0063] The vector can be an expression vector. In some embodiments, the vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a bacteriophage vector, etc.

[0064] (c) Recombinant cells containing the above-mentioned nucleic acid molecules and / or the above-mentioned recombinant vectors.

[0065] The recombinant cells are obtained by transforming or infecting host cells with the nucleic acid molecules or recombinant vectors of the present invention. In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is another cell suitable for preparing antibodies or their antigen-binding fragments.

[0066] In a third aspect, a method for preparing the antibody or antigen-binding fragment thereof described in this invention is provided, the method comprising culturing recombinant cells containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, in a manner suitable for expressing the antibody or antigen-binding fragment thereof.

[0067] In some embodiments, the method further includes: recovering the antibody or its antigen-binding fragment from the recombinant cells or culture medium.

[0068] In a fourth aspect, a composition is provided comprising the antibody or antigen-binding fragment thereof described in this invention or the biological material of this invention, and a pharmaceutically acceptable carrier.

[0069] In some embodiments, the above composition is a pharmaceutical composition.

[0070] In a fifth aspect, the use of the antibodies or antigen-binding fragments thereof, biological materials and / or compositions described herein in the preparation of any of the following products is provided:

[0071] (a) Products tested for LY6G6D;

[0072] (b) Products that stimulate or enhance the immune response;

[0073] (c) Products for the prevention and / or treatment of diseases associated with abnormal LY6G6D expression;

[0074] (d) Products that kill or inhibit the growth of cells expressing LY6G6D in vitro or in vivo.

[0075] In some implementations, the disease is colorectal cancer.

[0076] The antibody or its antigen-binding fragment provided by this invention binds to human LY6G6D and exhibits many superior properties, including the following:

[0077] 1. The antibody or its antigen-binding fragment provided by this invention binds to human LY6G6D with high affinity;

[0078] 2. It can trigger a specific anti-tumor immune response.

[0079] Therefore, the antibody or its antigen-binding fragment of the present invention has excellent drug development prospects. Attached Figure Description

[0080] Figure 1 The binding activity of the chimeric antibody against human LY6G6D to the human LY6G6D protein was measured.

[0081] Figure 2To assess the binding activity of the chimeric antibody against human LY6G6D to the LY6G6D protein of cynomolgus monkeys.

[0082] Figure 3 The chimeric antibody against human LY6G6D was tested for its binding activity with the HEK293 cell line expressing human LY6G6D.

[0083] Figure 4 To demonstrate the binding activity of the chimeric antibody against human LY6G6D to the cynomolgus monkey HEK293 cell line expressing LY6G6D.

[0084] Figure 5 The chimeric antibody against human LY6G6D was tested for its binding activity with endogenous human LY6G6D-expressing HT-55 cell lines.

[0085] Figure 6 To assess the binding activity of the chimeric antibody against human LY6G6D to the LS1034 cell line endogenously expressing human LY6G6D.

[0086] Figure 7 To assess the cytotoxic activity of human LY6G6D M014 CAR-T against HT-55 cell line.

[0087] Figure 8 To assess the cytotoxic activity of human LY6G6D M014 CAR-T against the LS1034 cell line.

[0088] Figure 9A This describes the changes in tumor volume during an in vivo anti-tumor experiment using anti-human LY6G6D M014 CAR-T. Figure 9B The changes in body weight during the in vivo anti-tumor experiment of anti-human LY6G6D M014 CAR-T. Detailed Implementation

[0089] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0090] Abbreviations and Definitions

[0091] Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have meanings known in the art.

[0092] "Antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by a ligand). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0093] "Antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0094] A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0095] The “Fc region” contains two heavy chain segments, including the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0096] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0097] A "single-chain Fv antibody" (or "scFv antibody") is an antibody fragment containing both VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. Generally, Fv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see U.S. Patent No. 6,423,538.

[0098] Those skilled in the art will understand that antibody heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region. Antibodies in the form of IgG1 are a subclass of IgG, with their heavy chain being the γ1 subtype. In some embodiments, the antibody disclosed in this invention is IgG1.

[0099] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this invention may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this invention comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies used in this invention may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant region can be modified to differ from naturally occurring immunoglobulin molecules in its amino acid sequence.

[0100] The "hypervariant region" refers to the antibody amino acid residues responsible for antigen binding. The hypervariant region contains the following amino acid residues: amino acid residues from the "complementarity-determining region" or "CDR" defined by sequence alignment. "Framework" residues or "FR" residues are variable domain residues other than those defined in the hypervariant region.

[0101] "Isolated antibody" is an antibody separated from all or part of its natural environmental components. The contaminating components of its natural environment are substances that could interfere with the diagnostic or therapeutic application of the antibody and may include enzymes, hormones, and other proteolysinic or non-proteolysinic substances. In some embodiments, the antibody is purified to the extent that: (1) more than 95% by weight, such as more than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a twist-cup sequencer; or (3) determined to be homogeneous by SDS-PAGE stained with Coomassie blue or silver under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. Isolated antibodies are typically prepared by at least one purification step. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0102] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0103] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0104] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, the individual antibodies constituting the group being identical. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0105] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0106] "Immune cells" include cells that have a hematopoietic origin and play a role in the immune response. Immune cells include: B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0107] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0108] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0109] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0110] As used herein, the term "about" means a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). Alternatively, "about" may mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable margin of error for that specific value.

[0111] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of a binding reaction of the protein, such as LY6G6D, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0112] When applied to polynucleotides, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is called "encoding" a polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0113] The term "identity" as used in this article can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0114] When "giving" and "treating" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treating" can refer to, for example, methods of treatment, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting a reagent with cells and contacting a reagent with a fluid, wherein the fluid contacts the cells. "Giving" and "treating" also mean, for example, in vitro and ex vivo treatment of cells by means of a reagent, diagnostic agent, conjugated composition, or other cells.

[0115] The term "treatment" refers to the improvement or cessation of a condition or its symptoms. Treatment includes suppression, such as reducing the overall frequency of attacks of a condition or its symptoms.

[0116] The term "prevention" includes avoiding the initial stages of a disease or its symptoms.

[0117] As used herein, the term "therapeutic effective dose" or "effective dose" refers to the amount of an anti-LY6G6D antibody or its antigen-binding fragment, administered alone or in combination with another therapeutic agent, that effectively prevents or alleviates the disease or condition to be treated. A therapeutic effective dose further refers to the amount of the antibody or its antigen-binding fragment sufficient to cause symptom relief, such relief being, for example, treatment, cure, prevention, or alleviation of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the symptom. The effective dose for a specific subject can vary depending on a variety of factors, such as the disease to be treated, the patient's overall health condition, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective dose refers to that single ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0118] Pharmaceutical Composition

[0119] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0120] In some implementations, the term "pharmaceuticalally acceptable carrier" refers to a substance approved by a government regulatory agency or listed in another recognized pharmacopoeia for use in animals (particularly for humans). Furthermore, "pharmaceuticalally acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0121] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with an active ingredient for therapeutic purposes. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier can be water when the drug composition is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, along with a suitable carrier, to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0122] In some embodiments, the pharmaceutical compositions of the present invention may be administered by any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration.

[0123] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The pharmaceutical composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

[0124] The antibodies or antigen-binding fragments thereof of the present invention include their salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0125] Example 1: Generation of mouse monoclonal antibody against human LY6G6D

[0126] 1. Obtaining single B-cell clones

[0127] Animal immunization: Human LY6G6D Protein, HIS Tag (Kaikai Biotechnology, catalog number: LYD-HM1GD) was used as an immunogen, diluted to 1 mg / mL with physiological saline, and then mixed with an equal volume of adjuvant CFA (Sigma, F5881) or IFA (Sigma, F5506) (CFA was used for the initial immunization, and IFA was used for subsequent immunizations). Balb / c and SJL female mice (6-8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with the protein via intraperitoneal injection (50 μg / mouse for the initial immunization, and 25 μg / mouse for each subsequent immunization). Another group of Balb / c mice received an initial immunization with CFA, and subsequent immunizations were administered using the Sigma Adjuvant System® Ribi (Sigma, S6322) via subcutaneous and intraperitoneal injection (50 μg / mouse for the initial immunization, and 25 μg / mouse for each subsequent immunization). Protein immunization was repeated at least three times with a two-week interval. Fourteen days after the last immunization, a second pulse immunization was performed by intraperitoneal injection of 25 μg of immunogen. Three days later, the spleen of the mice was harvested, and single CD138 cells were isolated and purified. +Single plasma cell (STEMCELL Tech; EasySep™ Mouse Pan-BCell Isolation Kit).

[0128] Single B-cell cloning: Utilizing a high-throughput single-cell optical fiber system (CaiKe Biotechnology; LyTARS) TM A high-throughput single-cell photoconductive system was used to screen for positive single-B cell clones that secrete anti-human LY6G6D specific antibodies. LyPartition TMA 14K single-cell screening chip (Cyco; catalog number: 81.07.0010) was placed into the LyTARS (Cyco LT-002) high-throughput single-cell photoconductive system chip platform. Chip wetting solution (Cyco; catalog number: 08.07.0010) and pure water were placed in the reagent compartment before the chip wetting process was run. After chip wetting, the chip initialization culture medium was used to replace the solution inside the chip and the system channels, filling the chip with culture medium. Enriched antibody-secreting cells (ACSs) were introduced into the chip in small volumes, using photoelectric tweezers to guide the single cells into the microcavities. The chip platform was set to 36°C to culture the cells, achieving the optimal temperature for ACS antibody secretion. The fluorescence detection mixture consisted of 400 μL of mouse plasma cell culture medium (Cyco; catalog number: 08.09.0010) and 1 μL of mouse IgG (Fc) fluorescent secondary antibody (AF647) (Cyco; catalog number: 08.10.0010). 50 μL of microspheres (Cyco; catalog number: 08.10.0010) were washed three times with PBS and reacted with biotinylated human LY6G6D protein (Kaikai Biotechnology, catalog number: LYD-HM4GDB) and biotinylated monkey LY6G6D protein (Kaikai Biotechnology, catalog number: LYD-CM16D), respectively. 7 μL of the fluorescence detection mixture was used to resuspend the antigen microspheres, and 7 μL of the detection microspheres were aspirated into the chip channels using a pump. The fluorescence scanning process was run, and the chip was scanned six times using the CY5 channel, with images acquired at 5-minute intervals. After scanning, the chip was rinsed three times with plasma cell culture medium to remove the detection microspheres from the flow channels. Cell lysis buffer (Cyco; catalog number: 08.06.0020) was prepared in advance and added at 5 μL per well to a 96-well PCR plate. The 96-well plate was placed in the well chamber, and export culture medium (Cyco; catalog number: 08.11.0010) was placed in the culture medium position of the reagent compartment. The single-cell export process was run, and plasma cells secreting anti-LY6G6D antibodies were pushed out of the microcavity using photoelectric tweezers and then exported to the 96-well PCR plate via the liquid flow system. Some of the plasma cell positive clones were sequenced. The corresponding chimeric antibody numbers and sequences of the positive single B cell clones are shown in Tables 1 and 2. The light and heavy chain variable regions obtained from sequencing (Table 2) were constructed onto the human constant region (IgG1 / K, Table 3) to construct the corresponding chimeric antibodies. The sequences were verified by sequencing for in vitro functional identification.

[0129] Table 1. Numbering of chimeric antibodies against human LY6G6D monoclonal B cells

[0130]

[0131] 2. Preparation of chimeric antibodies against human LY6G6D

[0132] Table 2. CDR and variable region sequences of chimeric antibodies against human LY6G6D (determined according to the Kabat protocol).

[0133]

[0134] The light and heavy chain variable regions (sequences shown in Table 2) were constructed onto the human constant region (IgG1 / K, sequence shown in Table 3) to construct the corresponding chimeric antibodies, and the sequences were verified by sequencing.

[0135] The corresponding nucleic acid encoding the chimeric antibody was expressed in Expi293 cells and purified using a Protein A column, as follows:

[0136] Expi293 cells expressing chimeric antibodies: One day before transfection, Expi293 cells (Thermo, catalog number: A14635CN) were diluted to a density of 1.5 × 10⁻⁶. 6 Cells / mL were cultured at 37°C in an 8% CO2 shaker at 120 rpm. On the second day, viable cell density and survival rate were measured; the cell transfection density should be 3 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%. Preparation of the PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, catalog number: 24765-1) by inverting. Dilute the expression plasmid (Invitrogen pCDNA3.4 vector, catalog number: A14697) with OPM-293CD05 medium (Shanghai Aopomai Biotechnology Co., Ltd., catalog number: 81075-001), to a total plasmid volume of 1 μg / mL. The volume of the medium used to dilute the plasmid should be 1 / 20 of the transfection volume. Mix gently, ensuring a light to heavy chain plasmid ratio of 1:1.5. Dilute the PEI reagent with OPM-293CD05 medium, to a volume 1 / 20 of the transfection volume. Mix gently by inverting and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and mix gently by inverting. Incubate the PEI / plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to a transfer flask, gently rotating the flask during the addition. After transfection, incubate the flask at 37°C and 8% CO2 on a shaker at 120 rpm. On the second day after transfection (24 hours post-transfection), add 10% OPM-293 ProFeed (Shanghai Aopumai Biotechnology Co., Ltd., catalog number: F081918) to the flask, gently rotating the flask during the addition. Then, return the flask to the shaker and continue incubating for 5-7 days, harvesting the supernatant.

[0137] Protein A column purification of antibodies: Prepare a gravity chromatography column. Open the column cap and place the gasket at the bottom of the column, pressing it firmly. Prepare the packing material, Protein A (Cytiva, catalog number: 17549801). Calculate the required packing suspension volume precisely based on the target packing volume and the packing suspension ratio: Required packing suspension volume = Target packing volume / Packing suspension ratio. Vortex the packing material thoroughly to ensure complete suspension. Add the packing suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the outlet pH. If the target pH of 7.4 is not reached, continue adding equilibration buffer until the target pH of 7.4 is reached. Slowly add a certain volume of sample to the column. Add at least 10 CV of eluent to the column. Slowly add 5CV of elution buffer (10-50 mM NaAc, pH 3.0-3.5) to the gravity chromatography column and incubate for 3-5 minutes. Collect the eluent. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH of 7.4 with neutralization buffer (1M Tris). Determine the protein concentration using Nanodrop. Replace the antibody-containing buffer with PBS via ultrafiltration.

[0138] Table 3. Constant region sequences and control antibodies

[0139]

[0140] Example 2: Functional identification of chimeric antibodies against human LY6G6D

[0141] Experiment 1: ELISA binding of anti-human LY6G6D chimeric antibody to human LY6G6D and cynomolgus monkey LY6G6D proteins.

[0142] The plate-coating proteins were human LY6G6D protein HIS Tag (Kaikai Biotechnology, catalog number: LYD-HM1GD) and cynomolgus monkey LY6G6D HIS Tag protein (Kaikai Biotechnology, catalog number: LYD-CM16D), 1 μg / mL, 100 μL / well, plated overnight at 4℃; 150 μL / well of 2% BSA was incubated at room temperature for 1 hour; 200 μL / well of PBST was washed three times; human LY6G6D chimeric antibody, positive control antibody hz20A12 (humanized 20A12 sequence in patent WO2021119505A1, whose heavy and light chain variable region amino acid sequences are shown as SEQ ID NO: 45 and 45, respectively, synthesized by Hongcheng Pharmaceutical), and negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were prepared into 900 μL of 33.333. nM stock solution; dilute the stock solution with blocking buffer at 3-fold dilutions, for a total of 8 gradients; add 100 μL / well of sample and incubate at room temperature for 1 hour; wash three times with PBST (containing 0.05% Tween 20) at 200 μL / well; add secondary antibody (goat anti-human Fab-HRP, Sigma, catalog number A0293-1ML, or goat anti-human IgG Fc-HRP, Sigma, catalog number A0170-1ML), diluted 1:5000, and incubate at 37℃ for 30 minutes; add 100 μL / well of secondary antibody and incubate at room temperature for 1 hour; wash three times with PBST (containing 0.05% Tween 20) at 200 μL / well; pat dry, add 100 μL of TMB chromogenic solution, develop for approximately 15 minutes, and then terminate the reaction with sulfuric acid. Read OD450 on the microplate reader. Data were analyzed using Graphpad Prism 8.0 software. The logarithm of antibody concentration was plotted on the x-axis, and the corresponding OD450 value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve, and the EC50 was calculated. The results are as follows: Figure 1 , Figure 2 As shown in Table 4.

[0143] The results showed that all anti-human LY6G6D chimeric antibodies had strong binding activity against human LY6G6D protein, comparable to the positive control antibody hz20A12. Among them, M001, M012, and M014 also had strong binding activity against cynomolgus monkey LY6G6D protein.

[0144] Table 4. Binding activity of anti-human LY6G6D chimeric antibody against human LY6G6D protein and cynomolgus monkey LY6G6D protein.

[0145]

[0146] Experiment 2: Binding activity of anti-human LY6G6D chimeric antibody with HEK293 cell lines overexpressing human LY6G6D and HEK293 cell lines overexpressing cynomolgus monkey LY6G6D.

[0147] The concentration of HEK293 cells expressing human LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27573) or HEK293 cell lines expressing cynomolgus monkey LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27574) was adjusted to 1×10 using FACS buffer (PBS solution containing 1% FBS). 6 Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The chimeric antibody against human LY6G6D, the positive control antibody hz20A12, and the negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were diluted to their initial working concentrations using FACS buffer (PBS solution containing 1% FBS), and then serially diluted using FACS buffer (as described in Experiment 1 above). The serially diluted antibodies were added to the wells at 100 μL / well to resuspend the cells, mixed thoroughly by pipetting, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, A-21445) was diluted 1:1000 using FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, the cell clumps were resuspended, mixed thoroughly by pipetting, and incubated at 4°C for approximately 45 minutes. After incubation, cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. Mean fluorescence intensity (MFI) was read using a flow cytometer (BDCelesta). The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 3 , 4 As shown in Table 5.

[0148] Table 5. Binding activity of anti-human LY6G6D chimeric antibody against human LY6G6D protein and cynomolgus monkey LY6G6D protein.

[0149]

[0150] The results showed that the anti-human LY6G6D chimeric antibodies M001, M012, and M014 had strong binding activity to cells expressing human LY6G6D and the HEK293 cell line expressing cynomolgus monkey LY6G6D.

[0151] Experiment 3: Binding activity of HT-55 cell line expressing endogenous human LY6G6D and LS1034 cell line expressing endogenous human LY6G6D.

[0152] The concentration of HT-55 cells (Nanjing Kebai, catalog number: CBP60012) and LS1034 cells (ATCC, catalog number: CRL2158ATC) expressing endogenous human LY6G6D was adjusted to 1×10⁻⁶ using FACS buffer (PBS solution containing 1% FBS). 6 Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The chimeric antibody against human LY6G6D, the positive control antibody hz20A12, and the negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were diluted to the initial working concentration using ACS buffer (PBS solution containing 1% FBS), and then serially diluted using FACS buffer (as described in Experiment 1 above). The serially diluted antibodies were added to the wells at 100 μL / well to resuspend the cells, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, catalog number: A-21445) was diluted 1:1000 using FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, the cell clumps were resuspended, mixed by pipetting, and incubated at 4°C for approximately 45 minutes. After incubation, cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. Mean fluorescence intensity (MFI) was read using a flow cytometer (BDCelesta). The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 5 , 6 As shown in Table 6.

[0153] Table 6. Binding activity of anti-human LY6G6D chimeric antibody against human LY6G6D protein and cynomolgus monkey LY6G6D protein.

[0154]

[0155] The results showed that the anti-human LY6G6D chimeric antibody M014 was comparable to the positive control anti-hz20A12 and had strong binding activity to the endogenous human LY6G6D cell lines HT-55 and LS1034.

[0156] Example 3: In vitro cell killing experiment of CAR-T cells

[0157] (1) Preparation of anti-LY6G6D CAR-T

[0158] The anti-LY6G6D antibody M014 and the positive control hz20A12 were used to construct the CAR structure. The full-length CAR consists of a CD8α signal peptide, an LY6G6D binding domain scFv (the heavy chain variable region is linked to the light chain variable region by 3 × G4S; the linker peptide is 3 × G4S), a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain from the N-terminus to the C-terminus. The amino acid and nucleotide sequences of each element of the CAR are shown in Table 7.

[0159] Table 7. Amino acid and nucleotide sequences of each CAR element

[0160]

[0161] (2) Lentiviral preparation

[0162] On day 0, HEK-293T cells (ATCC) were injected at a rate of 25,000 cells / cm³. 3 Inoculate into 10-layer cell factories, adding 1000 mL of DMEM medium containing 10% FBS to each factory; the next day, mix 200 μg PMD2.G, 200 μg PREV, 400 μg PRRE, and 800 μg PEV of the lentiviral packaging vector in 50 mL of DMEM (DNA mixture) and incubate at room temperature for 5 minutes; add 1.6 mL of PEI to 50 mL of DMEM, incubate at room temperature for 5 minutes, then add to the DNA mixture, incubate at room temperature for 15 minutes, and then add to the cell factories. Change the medium 6-8 hours after transfection. 48 hours after transfection, collect the viral supernatant, centrifuge to remove debris (4000g, 15 min), then centrifuge to concentrate (16000g, 4 h), remove the supernatant, resuspend in viral preservation solution, and aliquot.

[0163] (3) CAR-T preparation

[0164] On day 0, PBMCs (Shanghai Miaoshun, catalog number: P122051102C) were resuscitated, and T cells were sorted using DYNABEADs CD3 / CD28 (Gibco, 40203D). The purified T cells were activated in X-VIVO 15 (Lonza, BEBP02-054Q) supplemented with 5% FBS and 100 IU / mL IL-2 (RD, BT-002-GMP-050). T cells were re-seeded at 2E+05 cells / mL. Lentiviral virus was added to the T cells on day 2. On day 3, the culture medium was replaced with T cell expansion medium, i.e., X-VIVO 15 supplemented with 5% AB (GEMINI, 100-512) and 100 IU / mL IL-2 GMP. On day 5, DYNABEADs CD3 / CD28 were removed from the cell suspension using a magnetic pole, and the T cell expansion medium was replaced. On days 9 and 13, transduction efficiency was determined by detecting the percentage of T cells recognizing recombinant LY6G6D using flow cytometry. Cells were expanded using T cell expansion medium in larger culture vessels as needed. LY6G6D CAR-T cells were cryopreserved on day 14.

[0165] (4) CAR-T in vitro functional testing

[0166] Healthy luciferase-expressing target cells HT55 and LS1034 (1E+04 cells / well, 100 μL / well) were seeded into 96-well opaque white cell culture plates, with 3-5 replicates. The target-effect ratio was then calculated based on the CAR positivity rate, and effector cells (100 μL / well) were added at target-effect ratios of 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8, respectively. Culture medium was added as a negative control, and 2% Triton X-100 (BioFroxx, catalog number 1139ML100) was added as a maximum release control (MAX). After cell adhesion, effector cells were added. After co-incubation for 24h±2h, centrifuge at 125g for 5min, and aspirate 130μL of cell supernatant for cytokine detection. Add 70μL (1:1 with the liquid volume in the well plate) of pre-mixed glow-type firefly luciferase (Shanghai Yisheng, 11404ES80) to each well of the plate, shake well in the dark for 5-10min, detect the luciferescence signal, and calculate the CAR-T killing rate according to the following formula.

[0167] CAR-T kill rate (%) = (RLU_NC - RLU_(CAR-T)) / (RLU_NC - RLU_MAX) × 100%

[0168] The results of the in vitro killing ability test of HT-55 and LS1034 target cells endogenously expressing LY6G6D are as follows: Figure 7 , Figure 8 As shown, the CAR-T cells constructed from the candidate antibody M014 achieved superior in vitro killing activity compared to the positive control in HT-55 and LS1034 cells.

[0169] Example 4: In vivo functional testing of CAR-T cells

[0170] LS1034 (ATCC, catalog number: CRL2158ATC) mouse models were constructed to evaluate the in vivo antitumor effect of the candidate antibody M014 CAR-T. Frozen LS1034 cells were thawed, and the viable cell concentration was determined by cell counting. Cells were then seeded into culture dishes containing suitable culture medium for expansion culture. When cells entered the logarithmic growth phase, the cell suspension at the viable plateau was collected and mixed with matrix gel (CORNING, catalog number 354234) at a 1:1 volume ratio to prepare the inoculation solution, achieving a final cell density of 5E+06 cells / mL. The cells were subcutaneously injected into the experimental animals at a dose of 0.2 mL / animal. On day 8 post-inoculation, subcutaneous tumor growth parameters (major and minor axes) were monitored periodically using calipers, along with body weight. Tumor volume was calculated using the formula: volume = (minor axis^2 × major axis) / 2. The tumor volume was measured when it reached approximately 100 cm³. 3 Subsequently, the experimental animals were randomly assigned to different treatment groups; cell transplantation was performed via tail vein administration at a dose of 0.2 mL per animal, at a concentration of 5 × 10⁻⁶. 6 One CAR+ live cell / mouse; from the date of administration, tumor volume and body weight changes are continuously monitored at 3-4 day intervals until the experimental endpoint is reached.

[0171] The results are as follows Figure 9A and Figure 9B As shown, M014 CAR-T can significantly inhibit tumor growth in vivo, exhibiting excellent in vivo anti-tumor effects. Figure 9A Meanwhile, M014 CAR-T did not affect weight gain, indicating that M014 CAR-T was well tolerated.

Claims

1. An anti-LY6G6D antibody or its antigen-binding fragment, wherein, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:

26. The amino acid sequence of HCDR2 is shown in SEQ ID NO:

27. The amino acid sequence of HCDR3 is shown in SEQ ID NO:

28. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

30. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

31. The amino acid sequence of LCDR3 is shown in SEQ ID NO:

32.

2. The antibody or antigen-binding fragment thereof as described in claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO: 25; the amino acid sequence of the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:

29.

3. The antibody or antigen-binding fragment thereof as described in claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

29.

4. The antibody or antigen-binding fragment thereof as claimed in claim 3, further comprising a heavy chain constant region, a light chain constant region, or a combination thereof, or comprising an Fc region, a light chain constant region, or a combination thereof, wherein, The light chain constant region is a κ chain or λ chain constant region, and the heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD categories.

5. The antibody or antigen-binding fragment thereof as described in claim 4, wherein, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

34.

6. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, wherein, The antigen-binding fragments are F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv.

7. A CAR, from the N-terminus to the C-terminus, comprising, in sequence, a CD8α signal peptide, a heavy chain variable region, a linker peptide, a light chain variable region, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain; the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, the amino acid sequence of the linker peptide is shown in SEQ ID NO: 43, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29, the amino acid sequences of the CD8α hinge and the CD8α transmembrane region are shown in SEQ ID NO: 37, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 39, and the amino acid sequence of the partial CD3 intracellular domain is shown in SEQ ID NO:

41.

8. A biological material selected from (a) to (c) of the following. (a) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6 or a CAR as described in claim 7; (b) A recombinant vector comprising the nucleic acid molecule described in (a); (c) Recombinant cells comprising the nucleic acid molecules described in (a) and / or the recombinant vector described in (b).

9. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or a CAR according to claim 7, the method comprising, under conditions suitable for expressing the antibody or antigen-binding fragment thereof or CAR, culturing recombinant cells containing a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or a CAR according to claim 7.

10. A composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, or a CAR as described in claim 7, or a biological material as described in claim 8, and a pharmaceutically acceptable carrier.

11. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the CAR according to claim 7, the biomaterial according to claim 8, and / or the composition according to claim 10 in the preparation of any of the following products: (a) Products tested for LY6G6D; (b) Products for treating diseases associated with abnormal expression of LY6G6D; said disease is colorectal cancer.

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