Antibodies, antibody conjugates, and medications containing these antibodies.

Abstract

The invention is in the field of biomedical science. Specifically, the invention relates to an immunogenic polypeptide derived from the cTAGE5 protein, and also relates to an antibody that specifically binds to the immunogenic polypeptide.

Description

Antibodies and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention provides immunogenic polypeptides derived from cTAGE5 protein and applications thereof, and also provides antibodies that specifically bind to the immunogenic polypeptides and applications thereof.

[0002] Background of the Invention

[0003] Cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5), also known as MEA6 or MGEA6, is considered a tumor marker for various tumors, such as meningiomas, gliomas, and T-cell lymphomas. However, while cTAGE5 has been used as a tumor marker for tumor research, it has been discovered that cTAGE5 is also present in normal cells and is essential for cellular functions, such as participating in endoplasmic reticulum (ER) transport and export.

[0004] Previous studies investigating the function of cTAGE5 have primarily focused on the intracellular portion of cTAGE5 / Mea6. This study found that cTAGE5 / Mea6, consisting of 804 amino acid residues, forms a complex with TANGO1 on the ER membrane at the lumen, where it connects to the framework proteins Sec12, Sec22, and Sec23 of COPII, prompting COPII to export collagen. However, cTAGE5 / Mea6 does not leave the membrane but instead accompanies COPII. Similar studies have primarily investigated the behavior of cTAGE5 / Mea6 within the cell membrane, which is essential for the normal function of human cells. The amino acid sequence of the extracellular portion of cTAGE5 / Mea6, considered a signal peptide, has no known function.

[0005] Several commercial antibodies targeting cTAGE5 are available, such as Sigma-Aldrich products HPA000387 and HPA000922. However, these antibodies target the intracellular region of cTAGE5 / Mea6 (804 amino acids). Currently, there are no antibodies targeting the extracellular region of cTAGE5.

[0006] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1. Shows the sequence structure of the Mea6 splice variant of cTAGE5.

[0008] Figure 2 shows representative IHC results of rabbit polyclonal antibodies against ZZS01 and ZZS05.

[0009] Figure 3 shows some representative IHC results of 1D6 and F11 monoclonal antibodies.

[0010] Figure 4 shows that F11 monoclonal antibody significantly inhibits the growth of A-375 xenograft tumors in mice. A: Representative tumor morphology; B: Changes in mouse body weight; C: Changes in tumor volume. Results are expressed as mean ± SD (n = 8). **p < 0.01, significant difference compared to the control group.

[0011] Figure 5. Shows that F11 monoclonal antibody significantly inhibits HCC1937 xenograft tumor growth in mice. A: Representative tumor morphology; B: Changes in mouse body weight; C: Changes in tumor volume. Results are expressed as mean ± SD (n = 8). **p < 0.01, significant difference compared to the control group.

[0012] Figure 6. Humanized anti-ZZS05 antibody h32 mediates apoptosis in tumor organoids in an ADCC model. A: pancreatic cancer organoids; B: small cell lung cancer organoids.

[0013] Figure 7. Human anti-ZZS05 antibody h35 mediates apoptosis in different tumor organoids.

[0014] Figure 8. Human anti-ZZS05 antibody h35 mediated LDH release.

[0015] Detailed Description of the Invention

[0016] definition

[0017] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0018] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0019] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0020] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or completely synthetically (e.g., recombinantly), including any fragment thereof that retains the binding specificity of a full-length immunoglobulin comprising at least a portion of the variable region of an immunoglobulin molecule. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, camelid antibodies, single domain antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0021] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 M -1 -10 8 M -1 Antibodies to the Ka) antigen.

[0022] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies, which comprises antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0023] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by the fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell. As known to those of ordinary skill in the art, hybridomas can proliferate and continuously produce a specific monoclonal antibody. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0024] As used herein, a "conventional antibody" refers to an antibody comprising two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L'), and two antigen-binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include but are not limited to V and L'). H Chain, V H -C H 1 chain and V H -C H 1-C H 2-C H 3 chains), and each light chain can be a full-length light chain or any functional region (e.g., a light chain including but not limited to V L Chain and V L -C L Each heavy chain (H and H') is paired with a light chain (L and L', respectively).

[0025] As used herein, a full-length antibody is one having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L ) and hinge regions, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies having the same domains.

[0026] As used herein, dsFv refers to a fragment having a stable V H -V L Fv with engineered intermolecular disulfide bonds.

[0027] As used herein, a Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment having the same structure, for example, produced by recombinant synthesis.L and C L ) and another chain comprising a heavy chain variable region (V H ) and a constant region domain of the heavy chain (C H 1).

[0028] As used herein, a F(ab')2 fragment is an antibody fragment resulting from pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, for example, by recombinant methods. A F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain portion of which contains several additional amino acids, including cysteine, which forms a disulfide bond linking the two fragments.

[0029] As used herein, a Fab' fragment is a fragment comprising half (one heavy chain and one light chain) of a F(ab')2 fragment.

[0030] As used herein, a scFv fragment refers to a fragment comprising light chain variable regions (V L ) and heavy chain variable region (V H The length of the linker is such that the two variable domains are bridged without substantial interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues interspersed to increase solubility. n residue.

[0031] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0032] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0033] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to thereby improve one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their impact on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. In addition, the mutations in the CDRs are typically no more than one or two. Therefore, the humanized antibodies of the present invention also encompass antibodies comprising one or two amino acid mutations in the CDRs.

[0034] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0035] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region domain, VL (also denoted as V L ) and VH (also denoted as V L ). The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), which are part of the antigen binding site domain.

[0036] As used herein, "antigen binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A natural, conventional full-length antibody molecule has two conventional antigen-binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. A conventional antigen-binding site comprises loops connecting antiparallel beta chains within the variable region domain. An antigen-binding site may comprise other portions of the variable region domain. Each conventional antigen-binding site comprises three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. Hypervariable regions are also referred to as complementarity determining regions (CDRs).

[0037] As used herein, "hypervariable region," "HV," "complementarity determining region," "CDR," and "antibody CDR" are used interchangeably to refer to one of the multiple portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain comprises three CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises three CDRs, designated VL CDR1, VL CDR2, and VL CDR3 (or LCDR1, LCDR2, and LCDR3); the heavy chain variable region domain comprises three CDRs, designated VH CDR1, VH CDR2, and VH CDR3 (or HCDR1, HCDR2, and HCDR3). The three CDRs in the variable region are discontinuous along the linear amino acid sequence, but are proximal in the folded polypeptide. The CDRs are located within the loops that connect the parallel strands of the beta sheet of the variable domain. As described herein, those skilled in the art will recognize and can identify CDRs based on Kabat or Chothia numbering (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). Preferably, the CDRs of the present invention are numbered using the IMGT method. (2,3) For details, please refer to the method described in the embodiments of this article.

[0038] As used herein, framework regions (FRs) are domains within the antibody variable region domains that are located within the beta sheets; in terms of amino acid sequence, FR regions are relatively more conserved than hypervariable regions.

[0039] As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than the amino acid sequence of the variable region domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (C L ) domain, and each heavy chain comprises one or more heavy chain constant regions (C H ) domain, including C H 1. C H 2. C H 3 and C H 4. Full-length IgA, IgD, and IgG isotypes contain C H 1. C H 2. C H 3 and hinge regions, while IgE and IgM contain CH 1. C H 2. C H 3 and C H 4. C H 1 and C L The domains extend the Fab arms of the antibody molecule, thereby assisting in interacting with the antigen and rotating the antibody arms. The antibody constant region can serve effector functions such as, but not limited to, clearing the antigen, pathogen, and toxin to which the antibody specifically binds, for example, by interacting with various cells, biomolecules, and tissues.

[0040] As used herein, the functional region of an antibody is a region comprising at least VH, VL, C H (For example, C H 1. C H 2 or C H 3) C L or the hinge region domain or at least the functional region thereof.

[0041] As used herein, a functional region of a VH domain is at least a portion of an intact VH domain that retains at least part of the binding specificity of the intact VH domain (e.g., by retaining one or more CDRs of the intact VH domain), such that the functional region of the VH domain, alone or in combination with another antibody domain (e.g., VH domain), is a fully qualified antibody. L The functional region of an exemplary VH domain is a region comprising CDR1, CDR2 and / or CDR3 of the VH domain.

[0042] As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least some of the binding specificity of the complete VL domain (e.g., by retaining one or more CDRs of the complete VL domain), such that the functional region of the VL domain binds to the antigen alone or in combination with another antibody domain (e.g., a VH domain) or a region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of a VL domain.

[0043] As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present at a level of about 1 × 10 7 M -1 or 1x 10 8 M -1 or greater affinity constant Ka (or 1x 10-7 M or 1×10 -8 M or lower dissociation constant (K d )) binds to the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0044] As used herein, the term "competition" with respect to antibodies refers to a first antibody or its antigen-binding fragment binding to an epitope in a manner sufficiently similar to a second antibody or its antigen-binding fragment, whereby the binding result of the first antibody to its associated epitope is detectably reduced in the presence of the second antibody compared to the absence of the second antibody. Alternatively, in the case where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, this may be the case, but not necessarily so. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, in the case where each antibody can detectably inhibit the binding of another antibody to its associated epitope or ligand, whether to the same, higher or lower degree, the antibodies are referred to as "cross-competing" with each other in binding to their respective epitopes. Competition and cross-competing antibodies are encompassed in the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), those skilled in the art will recognize that such competition and / or cross-competing antibodies are encompassed in the present invention and can be used in the methods disclosed herein based on the teachings provided herein.

[0045] As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

[0046] An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody is (1) not associated with naturally associated components that accompany it in its native state, (2) free of other proteins from the same species, (3) expressed by cells from a different species, or (4) not occurring in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cells from which the polypeptide originates would be "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, i.e., using protein purification techniques well known in the art.

[0047] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0048] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by phosphodiester bonds.

[0049] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in the natural origin of nucleic acid molecules. " Isolated " nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesis. The exemplary isolated nucleic acid molecules provided herein comprise the isolated nucleic acid molecules of the antibody or Fab provided by coding.

[0050] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0051] As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

[0052] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0053] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0054] "Codon optimization" refers to a process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which is believed to be dependent on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See, Nakamura et al., 2001; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2029; 2030; 2040; 205 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0055] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0056] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0057] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0058] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided, as well as the compositions provided herein.

[0059] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0060] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0061] As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0062] As used herein, the term "subject" refers to a mammal, such as a human.

[0063] Polypeptide containing cTAGE5 extracellular region antigenic epitope and its application

[0064] In one aspect, the present invention provides an isolated immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the isolated polypeptide may further comprise a tag. The tag may be a tag for isolating and / or purifying the polypeptide, such as a His tag or a GST tag. The tag may also be a portion for enhancing the immunogenicity of the polypeptide.

[0065] In another aspect, the present invention provides a use of the isolated immunogenic polypeptide of the present invention in preparing an antibody. In some embodiments, the antibody is capable of specifically binding to the extracellular region of the cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2.

[0066] An exemplary cTAGE5 protein comprises the amino acid sequence of SEQ ID NO:415 or SEQ ID NO:416.

[0067] In another aspect, the present invention provides a method for preparing an antibody, the method comprising immunizing an animal with the isolated immunogenic polypeptide of the present invention, and then isolating an antibody that specifically binds to the polypeptide from the animal or obtaining sequence information of an antibody that specifically binds to the polypeptide. In some embodiments, the antibody is capable of specifically binding to the extracellular region of the cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2. In some embodiments, the animal can be any animal used in the art for preparing antibodies by immunization, including but not limited to mice, rats, rabbits, goats, alpacas, etc.

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising the isolated immunogenic polypeptide of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used to treat and / or prevent tumors. In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the vaccine further comprises an adjuvant.

[0069] In another aspect, the present invention provides use of the isolated immunogenic polypeptide of the present invention in the preparation of a medicament. In some embodiments, the medicament is used to treat and / or prevent tumors. In some embodiments, the medicament is a vaccine. In some embodiments, the vaccine further comprises an adjuvant.

[0070] In another aspect, the present invention provides a method for preventing and / or treating tumors in a subject in need thereof, comprising administering to the subject an effective amount of the isolated immunogenic polypeptide of the present invention or the pharmaceutical composition of the present invention.

[0071] In another aspect, the present invention provides a method for detecting the presence and / or amount of antibodies that specifically bind to a cTAGE5 protein in a biological sample, comprising 1) contacting the biological sample with an isolated immunogenic polypeptide of the present invention, and 2) detecting the presence and / or amount of antibodies that bind to the isolated immunogenic polypeptide. In some embodiments, the biological sample is a biological sample from a subject. In some embodiments, the subject is a subject suspected of having a tumor. In some embodiments, the biological sample is a tissue sample, a blood sample, or a lymphocyte sample. In some embodiments, the biological sample is a blood sample, such as whole blood, plasma, or serum. In some embodiments, the antibody specifically binds to the extracellular region of the cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence set forth in SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO:2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:2, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:2.

[0072] In another aspect, the present invention provides use of the isolated immunogenic polypeptide of the present invention in a kit for preparing a method for detecting the presence and / or amount of an antibody that specifically binds to a cTAGE5 protein in a biological sample. In some embodiments, the biological sample is a biological sample from a subject. In some embodiments, the subject is a subject suspected of having a tumor. In some embodiments, the biological sample is a tissue sample, a blood sample, or a lymphocyte sample. In some embodiments, the biological sample is a blood sample, such as whole blood, plasma, or serum. In some embodiments, the antibody specifically binds to the extracellular region of the cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence set forth in SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO:2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:2, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:2.

[0073] On the other hand, the present invention provides the use of a cTAGE5 protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, preferably comprising the amino acid sequence shown in SEQ ID NO: 2, as a tumor marker.

[0074] In another aspect, the present invention provides a method for tumor screening, comprising detecting the presence or amount of cTAGE5 protein in a biological sample from a subject, wherein the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, preferably comprises the amino acid sequence shown in SEQ ID NO: 2.

[0075] In another aspect, the present invention provides a method for tumor screening, which comprises detecting the presence and / or amount of an antibody that specifically binds to a cTAGE5 protein in a biological sample from a subject, wherein the antibody specifically binds to the extracellular region of the cTAGE5 protein, and the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, preferably comprises the amino acid sequence shown in SEQ ID NO: 2.

[0076] In some embodiments of the various aspects of the present invention, the tumor is a cTAGE5 protein-associated tumor, such as a tumor that overexpresses the cTAGE5 protein. In some embodiments, the cTAGE5 protein has an extracellular region. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2. Examples of the tumor include, but are not limited to, nasopharyngeal carcinoma, malignant melanoma, lung cancer, glioma, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, endometrial cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer (such as triple-negative breast cancer), esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, and colorectal cancer. In some embodiments, the tumor is adenocarcinoma. In some embodiments, the tumor is squamous cell carcinoma. In some embodiments (e.g., the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO: 1), the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung squamous cell carcinoma, cervical cancer, skin cancer, glossopharyngeal carcinoma, and esophageal squamous cell carcinoma. In some embodiments (for example, the extracellular region of cTAGE5 comprises the amino acid sequence shown in SEQ ID NO: 2), the tumor is selected from B-cell lymphoma, T-cell lymphoma, malignant melanoma, lung squamous cell carcinoma, lung adenocarcinoma, liver cancer, cervical squamous cell carcinoma, thyroid cancer, colon adenocarcinoma, ovarian cancer, brain tumor, skin squamous cell carcinoma, prostate cancer, breast cancer (such as triple-negative breast cancer), esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastric cancer, pancreatic cancer, rectal cancer, endometrial cancer, and colorectal cancer.

[0077] Antibodies against the extracellular region of cTAGE5

[0078] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of a cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2. In some embodiments, the antibody is a polyclonal antibody. In some preferred embodiments, the antibody is a monoclonal antibody.

[0079] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to an immunogenic polypeptide, wherein the immunogenic polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antibody is a polyclonal antibody. In some preferred embodiments, the antibody is a monoclonal antibody.

[0080] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody comprises the heavy chain CDRs and light chain CDRs, and / or the heavy chain variable region and light chain variable region of any one of the antibodies in Tables 2 and 6.

[0081] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and comprises a heavy chain CDR and a light chain CDR selected from any one of the following groups:

[0082] In some embodiments, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region (VH) and the light chain variable region (VL) each comprise an amino acid sequence selected from any one of the following groups or each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with an amino acid sequence selected from any one of the following groups:

[0083] In some embodiments, the monoclonal antibody is a humanized antibody. In some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30.

[0084] In some embodiments, the monoclonal antibody comprises a heavy chain constant region set forth in SEQ ID NO: 417. In some embodiments, the monoclonal antibody comprises a light chain constant region set forth in SEQ ID NO: 418. In some embodiments, the monoclonal antibody comprises a heavy chain constant region set forth in SEQ ID NO: 417 and a light chain constant region set forth in SEQ ID NO: 418.

[0085] In some embodiments, the monoclonal antibody comprises the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4. In some embodiments, the monoclonal antibody comprises the heavy chain of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14.

[0086] In some embodiments, the monoclonal antibody specifically binds to the extracellular region of the cTAGE5 protein. In some embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0087] In some embodiments, the monoclonal antibody specifically binds to an immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0088] In one aspect, the present invention further comprises a monoclonal antibody or antigen-binding fragment thereof that can compete for binding to the extracellular region of a cTAGE5 protein with a monoclonal antibody consisting of a heavy chain of SEQ ID NO: 3 and a light chain of SEQ ID NO: 4, or with a monoclonal antibody consisting of a heavy chain of SEQ ID NO: 13 and a light chain of SEQ ID NO: 14. In some embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence of SEQ ID NO: 2.

[0089] In one aspect, the present invention further comprises a monoclonal antibody or an antigen-binding fragment thereof that can compete with a monoclonal antibody consisting of a heavy chain shown in SEQ ID NO: 3 and a light chain shown in SEQ ID NO: 4, or with a monoclonal antibody consisting of a heavy chain shown in SEQ ID NO: 13 and a light chain shown in SEQ ID NO: 14 for binding to an immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0090] Antibodies of the present invention, such as monoclonal antibodies of the present invention or antigen-binding fragments thereof, are capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%.

[0091] The K-binding activity of the antibody of the present invention, such as the monoclonal antibody or antigen-binding fragment thereof, which binds to the extracellular region of the cTAGE5 protein (e.g., the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2) or the isolated immunogenic polypeptide of the present invention (e.g., the immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2). D The value can be less than about 1×10 -7 M, preferably less than about 1×10 -8 M, more preferably less than about 1×10 -9 M, more preferably less than about 1×10 -10 M.

[0092] Nucleic acids, vectors, and antibody production methods

[0093] On the other hand, the present invention provides isolated nucleic acid molecules, which encode the aforementioned antibodies of the present invention or their Fabs. In some embodiments, the nucleotide sequence of the nucleic acid molecules is codon-optimized for the host cell for expression. In some embodiments, the nucleic acid molecules of the present invention are operably connected to an expression control sequence.

[0094] The present invention also provides an expression vector comprising at least one of the aforementioned nucleic acid molecules of the present invention.

[0095] The present invention also provides a host cell transformed with at least one of the aforementioned nucleic acid molecules or expression vectors of the present invention.

[0096] In another aspect, the present invention provides a method for producing an antibody or antigen-binding fragment thereof of the present invention, comprising:

[0097] (i) culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and

[0098] (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the host cell.

[0099] The present invention also relates to an isolated antibody or antigen-binding fragment thereof obtained by the above-mentioned method of the present invention, which is capable of specifically binding to the extracellular region of the cTAGE5 protein. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2.

[0100] Antibody conjugates

[0101] The present invention also provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof of the present invention and a therapeutic moiety conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic moiety includes, for example, a cytotoxin, a radioisotope, or a biologically active protein.

[0102] Cytotoxins include any agent that is detrimental to cells (e.g., kills cells). Examples include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0103] Therapeutic moieties useful for conjugation also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthramycins (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin D (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0104] Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies or fragments thereof of the present invention include duocarmycins, calicheamicins, maytansines, auristatins, and derivatives thereof.

[0105] Cytotoxins can be conjugated to the antibodies of the present invention using linker technology used in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Linkers can be selected that are, for example, easily cleaved by low pH within the lysosomal compartment or that are easily cleaved by proteases, such as proteases preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D).

[0106] The antibodies of the present invention can also be conjugated to radioactive isotopes to produce cytotoxic radiopharmaceuticals, also known as radioactive antibody conjugates. Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioactive antibody conjugates are well established in the art.

[0107] The antibodies of the present invention can also be conjugated to proteins with desired biological activities and can be used to modify specific biological responses. Such biologically active proteins include, for example, toxins or active fragments thereof with enzymatic activity, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins, such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), interleukin-10 ("IL-10"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other immune factors such as IFN.

[0108] Disease treatment and / or prevention

[0109] Thus, the present invention provides a method for preventing and / or treating tumors in a subject, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof of the present invention or the antibody conjugate of the present invention.

[0110] The tumor that can be treated and / or prevented by the method of the present invention is a cTAGE5 protein-associated tumor, such as a tumor that overexpresses the cTAGE5 protein. In some embodiments, the cTAGE5 protein has an extracellular region. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2.

[0111] Examples of the tumors that can be treated and / or prevented by the methods of the present invention include, but are not limited to, nasopharyngeal carcinoma, malignant melanoma, lung cancer, glioma, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer (such as triple-negative breast cancer), esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, endometrial cancer, and colorectal cancer. In some embodiments, the tumor is adenocarcinoma. In some embodiments, the tumor is squamous cell carcinoma. In some embodiments (e.g., the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO: 1), the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung squamous cell carcinoma, cervical cancer, skin cancer, glossopharyngeal carcinoma, and esophageal squamous cell carcinoma. In some embodiments (for example, the extracellular region of cTAGE5 comprises the amino acid sequence shown in SEQ ID NO: 2), the tumor is selected from B-cell lymphoma, T-cell lymphoma, malignant melanoma, lung squamous cell carcinoma, lung adenocarcinoma, liver cancer, cervical squamous cell carcinoma, thyroid cancer, colon adenocarcinoma, ovarian cancer, brain tumor, skin squamous cell carcinoma, prostate cancer, breast cancer (such as triple-negative breast cancer), esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastric cancer, pancreatic cancer, rectal cancer, endometrial cancer, and colorectal cancer.

[0112] Pharmaceutical composition

[0113] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the antibody conjugate of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition is used for preventing and / or treating tumors in a subject.

[0114] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0115] The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0116] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0117] Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. In many cases, it is preferred to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, or sodium hydroxide in the composition. Prolonged absorption of injectable drugs can be achieved by including agents that delay absorption, such as monostearate and gelatin in the composition.

[0118] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except to the extent that they are incompatible with the active compound, may be included in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

[0119] Therapeutic compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained.

[0120] Sterile injections can be prepared by mixing the active compound in a suitable solvent in the required amount and adding one or a combination of the ingredients listed above as needed, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injections, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), from which a powder of the active ingredient plus any additional required ingredients is obtained from a previously sterile-filtered solution thereof.

[0121] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0122] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specific specification of the dosage unit form of the present invention is limited by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art for formulating such active compound for treating individual sensitivity.

[0123] For the administration of antibody molecules, dosage range is about 0.0001 to 100 mg / kg, more generally 0.01 to 20 mg / kg recipient body weight. For example, dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or within the range of 1-20 mg / kg. Exemplary treatment regimens require weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or initial dosing interval slightly shorter (such as once a week to once every three weeks) later dosing interval lengthening (such as once a month to once every 3-6 months).

[0124] Alternatively, the antibody molecule can also be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. Generally, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are given at less frequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, higher dosages need to be given at shorter intervals sometimes until the progression of the disease is alleviated or stopped, preferably until the patient shows partial or complete improvement in symptoms of the disease. Afterwards, the patient can be administered with a preventive regimen.

[0125] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention being employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of treatment, other drugs, compounds, and / or materials being used in combination with the particular composition being employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0126] An "effective amount" of an antibody or antigen-binding fragment thereof, or an antibody conjugate of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevents damage or disability caused by the suffering of the disease. For example, for the treatment of tumors, an "effective amount" of an antibody or antigen-binding fragment thereof of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that is predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by assays known to those skilled in the art. An effective amount of an antibody or antigen-binding fragment thereof of the invention is capable of reducing tumor size or otherwise alleviating symptoms in a subject, such as preventing and / or treating metastasis or recurrence. Such an amount can be determined by those skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0127] The antibodies of the present invention or their antigen-binding fragments, the antibody conjugates of the present invention or the pharmaceutical compositions of the present invention can be administered by one or more routes of administration using one or more methods well known in the art. It will be understood by those skilled in the art that the route of administration and / or mode vary depending on the desired result. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion, or bladder instillation. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0128] Alternatively, the antibodies or antigen-binding fragments thereof of the invention, or the antibody conjugates of the invention, or the pharmaceutical compositions of the invention can also be administered non-parenterally, such as topically, epidermally or mucosally, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0129] The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0130] In certain embodiments, the antibodies of the present invention can be formulated to ensure proper distribution in the body. For example, the blood-brain barrier (BBB) ​​blocks many highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the present invention can cross the BBB (if necessary), they can be formulated in, for example, liposomes.

[0131] Chimeric Antigen Receptor (CAR)

[0132] The antibodies or antigen-binding fragments thereof of the present invention can also be used to prepare chimeric antigen receptors (CARs), which, after being introduced into T cells or NK cells, can be used for cell therapy of related tumors (CAR-T or CAR-NK therapy). Therefore, the present invention also covers chimeric antigen receptors (CARs) comprising antibodies or antigen-binding fragments thereof of the present invention, CAR-T or CAR-NK cells comprising the CAR, and the use of the CAR-T or CAR-NK cells in treating tumors.

[0133] Combination therapy

[0134] The polypeptides, antibodies or antigen-binding fragments thereof, antibody conjugates or pharmaceutical compositions of the present invention can be administered in combination with chemotherapeutic agents or antibodies targeting other tumor antigens.

[0135] There are no particular limitations on the chemotherapeutic agents or antibodies targeting other tumor antigens used in combination with the polypeptides, antibodies, or antigen-binding fragments thereof, antibody conjugates, or pharmaceutical compositions of the present invention. Examples of the chemotherapeutic agents and antibodies targeting other tumor antigens include, but are not limited to, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, doxifluridine, nelarabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin , daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cisplatin, nedaplatin, anastrozole, exemestane, ethinyl estradiol, chlormadinone acetate, goserelin, tamoxifen, dexamethasone, bicalutamide, toremifene, flutamide, prednisolone, fosfastol, mitotane, methyltestosterone, leuprolide, letrozole, methylprogesterone acetate, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, and lapatinib.

[0136] The polypeptide, antibody or antigen-binding fragment thereof, antibody conjugate of the present invention and the chemotherapeutic agent or antibody targeting other tumor antigens can be all administered at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0137] The combined dosage of the polypeptide, antibody, or antigen-binding fragment thereof, antibody conjugate of the present invention and the chemotherapeutic agent or antibody targeting other tumor antigens in the pharmaceutical composition of the present invention is not particularly limited. As described above, the dosage of the antibody of the present invention can be determined by reference to the dosage when the antibody is used alone. The chemotherapeutic agent and antibody targeting other tumor antigens can be used according to the dosage indicated for each drug or can be reduced (taking into account the combined effect with the antibody of the present invention).

[0138] The polypeptides, antibodies or antigen-binding fragments thereof, antibody conjugates or pharmaceutical compositions of the present invention can also be combined with radiotherapy, for example, comprising administering ionizing radiation to the patient before, during and / or after administration of the antibody or pharmaceutical composition of the present invention.

[0139] Detection and diagnosis

[0140] In another aspect, the present invention also provides a method for detecting the presence of a cTAGE5 protein or the expression level of a cTAGE5 protein in a biological sample, comprising contacting the biological sample and, optionally, a control sample, with an antibody, such as a monoclonal antibody or its antigen-binding fragment, under conditions capable of forming a complex between the antibody, such as a monoclonal antibody or its antigen-binding fragment, and the cTAGE5 protein. The formation of the complex is then detected, wherein the formation of the complex in the biological sample or the difference in complex formation between the biological sample and the control sample indicates the presence of the cTAGE5 protein or the expression level of the cTAGE5 protein in the sample. In some embodiments, the cTAGE5 protein has an extracellular region. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO:2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:2, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:2.

[0141] In some embodiments, the control biological sample is a biological sample from a healthy individual. In some embodiments, the control biological sample is a biological sample from an individual known not to have a tumor.

[0142] In some embodiments, the biological sample is an in vitro sample. The biological sample includes but is not limited to a tissue sample, a blood sample, a lymph sample, and the like.

[0143] In some embodiments, the biological sample is a tissue section. In some embodiments, the method is an immunohistochemical assay. In some embodiments, the method is an immunofluorescence assay.

[0144] The present invention surprisingly discovered that cTAGE5 comprising the amino acid sequence of SEQ ID NO:1 is highly expressed in malignant melanoma and squamous cell carcinoma, while cTAGE5 comprising the amino acid sequence of SEQ ID NO:2 is highly expressed in most common tumors. Therefore, the present invention's antibodies, such as monoclonal antibodies or antigen-binding fragments thereof, can be used to screen for or diagnose tumors associated with cTAGE5.

[0145] Therefore, the present invention also provides a method for detecting the presence of a tumor (e.g., tumor cells) in a patient, comprising detecting (e.g., detecting by the method of the present invention) the presence or expression level of a cTAGE5 protein in a biological sample from the patient, wherein the presence of cTAGE5 in the patient's biological sample or the expression level of cTAGE5 in the patient's biological sample is higher than the expression level of cTAGE5 in a control biological sample, indicating the presence of a tumor (e.g., tumor cells) in the patient. In some embodiments, the cTAGE5 protein has an extracellular region. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2. In some embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some preferred embodiments, the extracellular region of the cTAGE5 protein comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the method can also indirectly detect the presence or expression level of the cTAGE5 protein by detecting the presence and / or amount of an mRNA molecule encoding the cTAGE5 extracellular region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2).

[0146] In some embodiments, the control biological sample is a biological sample from a healthy individual. In some embodiments, the control biological sample is a biological sample from an individual known not to have a tumor.

[0147] In some embodiments, the biological sample is an in vitro sample. The biological sample includes but is not limited to a tissue sample, a blood sample, a lymph sample, and the like.

[0148] In some embodiments, the biological sample is a tissue section. In some embodiments, the method is an immunohistochemical assay. In some embodiments, the method is an immunofluorescence assay.

[0149] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing cTAGE5-related diseases, such as tumors, comprising an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof, of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the diagnostic agent is a contrast agent.

[0150] In another aspect, the present invention provides use of an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof, of the present invention in the preparation of a diagnostic agent for detecting and / or diagnosing a cTAGE5-related disease, such as a tumor. In some embodiments, the diagnostic agent is a contrast agent.

[0151] In another aspect, the present invention provides a method for detecting and / or diagnosing a cTAGE5-related disease, such as a malignant tumor, in a subject, comprising administering to the subject an antibody, such as a monoclonal antibody or antigen-binding fragment thereof, or a diagnostic agent, of the present invention.

[0152] In some embodiments of the above aspects of the invention, the antibodies, monoclonal antibodies or antigen-binding fragments thereof of the invention are further conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, labels, etc. that can be used for detection or can be detected by other reagents.

[0153] In some embodiments of the various aspects of the present invention, the tumor is a cTAGE5 protein-associated tumor, such as a tumor that overexpresses the cTAGE5 protein. In some embodiments, the cTAGE5 protein has an extracellular region. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1. In some embodiments, the cTAGE5 extracellular region comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 2. Examples of the tumor include, but are not limited to, nasopharyngeal carcinoma, malignant melanoma, lung cancer, glioma, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, and rectal cancer. In some embodiments, the tumor is adenocarcinoma. In some embodiments, the tumor is squamous cell carcinoma. In some embodiments (e.g., the cTAGE5 extracellular region comprises the amino acid sequence shown in SEQ ID NO: 1), the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung squamous cell carcinoma, cervical cancer, skin cancer, glossopharyngeal carcinoma, and esophageal squamous cell carcinoma. In some embodiments (for example, the extracellular region of cTAGE5 comprises the amino acid sequence shown in SEQ ID NO: 2), the tumor is selected from B-cell lymphoma, T-cell lymphoma, malignant melanoma, lung squamous cell carcinoma, lung adenocarcinoma, liver cancer, cervical squamous cell carcinoma, thyroid cancer, colon adenocarcinoma, ovarian cancer, brain tumor, skin squamous cell carcinoma, prostate cancer, breast cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastric cancer, pancreatic cancer, and rectal cancer.

[0154] Reagent test kit

[0155] Also within the scope of the present invention are kits for use in the methods of the present invention, comprising a polypeptide, an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, or an antibody conjugate of the present invention, and instructions for use. The kit may further comprise at least one additional detection reagent for detecting the presence of the polypeptide or monoclonal antibody of the present invention. Kits generally include a label indicating the intended use and / or instructions for use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise accompanying the kit. Example

[0156] A further understanding of the present invention may be obtained by reference to some of the specific embodiments provided herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Obviously, various modifications and variations may be made to the present invention without departing from the spirit of the present invention, and therefore, such modifications and variations are also within the scope of the present application.

[0157] IHC experimental methods and procedures

[0158] 1) Bake the slides at 60°C for 30 minutes and dewax and hydrate as usual;

[0159] 2) Antigen retrieval: Antigen retrieval was performed with 0.01 M citric acid buffer (pH 6.0) at high pressure for 2 minutes, cooled to room temperature, and washed with phosphate buffered saline (PBS) for 5 minutes x 3 times

[0160] 3) Block endogenous peroxidase with 3% H202-methanol at room temperature for 10 minutes, and wash with PBS for 5 minutes × 3 times;

[0161] 4) Add normal non-immune animal serum dropwise and incubate at room temperature for 10 minutes;

[0162] 5) Remove serum and add primary antibody (polyclonal antibody or ID6 or F11) dilution solution and refrigerate at 4°C overnight;

[0163] 6) Wash with 0.1% Tween-20 PBS for 5 minutes x 3 times;

[0164] 7) Add biotin-labeled goat anti-mouse / rabbit IgG and incubate at room temperature for 10 minutes;

[0165] 8) Wash with 0.1% Tween-20 PBS for 5 minutes x 3 times;

[0166] 9) Add streptavidin-peroxidase dropwise and incubate at room temperature for 10 minutes;

[0167] 10) DAB color development for 5 minutes, followed by washing with distilled water to terminate color development;

[0168] 11) Hematoxylin counterstaining, water washing, differentiation and then thorough water washing to return to blue;

[0169] 12) Conventional dehydration and transparency, neutral gum sealing.

[0170] Example 1: Identification of the extracellular region of cTAGE5

[0171] The cTAGE5 gene has more than 20 exons, and due to alternative transcription, 28 cTAGE5 splice variants are present in the database. The proteins of the 28 cTAGE5 splice variants are summarized in Table 1.

[0172] Table 1. The intra- and extra-membrane structures of 28 cTAGE5 splice variants. Eight variants lack a transmembrane region, another eight variants contain a short peptide with the same sequence outside the transmembrane region, and another 12 variants have an extra-membrane region that extends beyond the extra-membrane peptides of the eight variants and extends further toward the N-terminus.

[0173] Previous studies on the transport protein function of cTAGE5 have primarily focused on the Mea6 variant of cTAGE5, also known as Mgea6. This is one of the 12 splice variants with a longer peptide outside the transmembrane region. Its structure is roughly shown in Figure 1.

[0174] Previous studies investigating the function of cTAGE5 have primarily focused on the intracellular portion of cTAGE5 / Mea6. This study found that cTAGE5 / Mea6, consisting of 804 amino acid residues, forms a complex with TANGO1 on the ER membrane at the lumen, where it connects to the framework proteins Sec12, Sec22, and Sec23 of COPII, prompting COPII to export collagen. However, cTAGE5 / Mea6 does not leave the membrane but instead accompanies COPII. Similar studies have primarily investigated the behavior of cTAGE5 / Mea6 within the cell membrane, which is essential for the normal function of human cells. The amino acid sequence of the extracellular portion of cTAGE5 / Mea6, considered a signal peptide, has no known function.

[0175] The 12 splice variants with longer peptides outside the transmembrane region contain three different N-terminal extension sequences: 9 variants have the N-terminal extension sequence: MEEPGVTPQPYLGLLLEELRRVVAALPEG (SEQ ID NO: 1); the other two variants have the N-terminal long sequence: MELKSPEEEVVAALPEG (SEQ ID NO: 2); the extension sequence of another variant has no immunogenicity and is not considered here.

[0176] Herein, the sequence MEEPGVTPQPYLGLLLEELRRVVAALPEG (SEQ ID NO: 1) is designated as ZZS01; the sequence MELKSPEEEVVAALPEG (SEQ ID NO: 2) is designated as ZZS05.

[0177] The amino acid sequence of an exemplary cTAGE5 protein containing ZZS01 (cTAGE5 variant 1; Protein_id="NP_005921.2") is as follows:

[0178] The amino acid sequence of an exemplary cTAGE5 protein comprising ZZS05 (cTAGE5 variant 2; Protein_id="NP_976229.1") is as follows:

[0179] Example 2. Polyclonal antibody preparation and immunohistochemistry experiments

[0180] The coding sequences of ZZS01 and ZZS05 were inserted into the vector pGEX-6p-1, and the GST-ZZS01 and GST-ZZS05 fusion proteins were expressed in BL21 E. coli and purified. Rabbits were immunized with these two fusion proteins, and rabbit sera against ZZS01 and ZZS05 were harvested.

[0181] The resulting rabbit serum was used to perform immunohistochemistry on tissue sections from over 20 common tumors. The results showed that the ZZS01 polyclonal antibody was sensitive to malignant melanoma and squamous cell carcinoma (but not adenocarcinoma), while the ZZZ05 polyclonal antibody was sensitive to all of these tumors. Representative IHC results using these rabbit polyclonal antibodies are shown in Figure 2.

[0182] Example 3. Preparation of mouse monoclonal antibodies and immunohistochemistry experiments

[0183] 3.1 Preparation of Mouse Monoclonal Antibodies

[0184] GST-ZZS01 and GST-ZZS05 fusion proteins were used to prepare mouse monoclonal antibodies. ZZS01 and ZZS05 polypeptides were used to select hybridoma cells expressing specific antibodies. Total RNA was extracted from hybridoma cells expressing high levels of the antibodies, and the mRNA sequences of the antibodies were obtained using RT-PCR. The two antibodies, along with their original signal peptides, were inserted into pCHO1.0 and pcDNA3.1 vectors for expression in CHO cells. The expressed cell culture medium was centrifuged and then applied to a Protein-G affinity column. The antibodies were eluted with a pH 2.6 glycine solution and further purified using an ion exchange resin. The antibodies, concentrated with an appropriate buffer, were placed in a refrigerator (-20°C) for later use.

[0185] The sequence information of the obtained mouse monoclonal antibody is shown in Table 2.

[0186] Table 2. Mouse monoclonal antibody sequence information (CDRs are underlined)

[0187] 3.2 Immunohistochemistry

[0188] As shown in Tables 3 and 4 below, immunohistochemical examinations were performed on pathological tissue sections of tumor patients using 1D6 antibody and F11 antibody, respectively.

[0189] Table 3. Patient pathological tissue block information for 1D6 antibody IHC

[0190] TNM: Tumor clinical staging system, T, N, M represent the size of the primary tumor, regional lymph node metastasis, and the presence of distant metastasis (Tumor, Node, Metastasis), respectively.

[0191] Table 4. Patient pathological tissue paraffin block information for F11 antibody used in IHC

[0192] TNM: Tumor clinical staging system, T, N, M represent the size of the primary tumor, regional lymph node metastasis, and the presence of distant metastasis (Tumor, Node, Metastasis), respectively.

[0193] The results showed that the specificity of monoclonal antibodies 1D6 and F11 for tumor cell recognition was consistent with the results of the polyclonal antibodies in Example 2. Antibody 1D6 was able to specifically recognize malignant melanoma and squamous cell carcinoma (including all tumors listed in Table 3: nasopharyngeal carcinoma, malignant melanoma, lung squamous cell carcinoma, cervical cancer, skin cancer, glossopharyngeal carcinoma, and esophageal squamous cell carcinoma); while F11 was able to specifically recognize all tested tumors (tumors listed in Table 4: B-cell lymphoma, T-cell lymphoma, malignant melanoma, lung squamous cell carcinoma, lung adenocarcinoma, liver cancer, cervical squamous cell carcinoma, thyroid cancer, colon adenocarcinoma, ovarian cancer, brain tumor, skin squamous cell carcinoma, prostate cancer, breast cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastric cancer, pancreatic cancer, and rectal cancer). At the same time, these antibodies did not bind to adjacent tissues (normal cells). Representative immunohistochemistry results are shown in Figure 3.

[0194] These results suggest that the cTAGE5 protein containing ZZS01 is overexpressed in human malignant melanoma and squamous cell carcinoma tissue cells, while the cTAGE5 protein containing ZZS05 is overexpressed in all tumor tissue cells in the tumor immunohistochemistry experiments performed. Therefore, ZZS01 is a specific epitope for human malignant melanoma and squamous cell carcinoma tissue cells, and ZZS05 is a specific epitope for human tumor tissue cells.

[0195] 3.3. Mouse Antibody Affinity Determination

[0196] The antigen binding affinity of the monoclonal murine antibodies 1D6 or F11 was determined by Biacore using antigens ZZS01 and ZZS05, respectively.

[0197] General process:

[0198] 1. Preparation of mobile phase buffer: dilute 10x HBS-EB + buffer to 10x;

[0199] 2.25℃ test, mobile phase is HBS-EP + buffer;

[0200] 3. Inject the antigen into the sensor chip CM5 coated with goat anti-GST antibody to fix the GST-antigen fusion protein;

[0201] 4. Diluted antibodies are injected into the channel of the chip as the binding phase, and then the mobile phase is injected as the dissociation phase.

[0202] Experimental setup:

[0203] The result is as follows:

[0204] 3.4 Humanization of mouse antibodies

[0205] NIH Protein Blast was used to search for amino acid sequences of human antibodies VH and VL in Protein Data Bank (PDB) that were close to 1D6 or F11. After comparing the amino acid sequences, the corresponding spatial structures were used as templates for 1D6 VH, 1D6 VL, F11 VH, and F11 VL. (5) Using WinCoot, a model building and structure display software, the amino acid sequences in the PDB that differed from their respective amino acids were replaced to construct the spatial structure models of 1D6 VH, 1D6 VL, F11 VH, and F11 VL. The VH and VL of each antibody were aligned with the relative positions in the PDB and assembled to form a 3D spatial model of the variable region. (6) The lowest energy extreme value calculation of the structure was performed to obtain the computer-simulated spatial structure model of the 1D6 variable region and the F11 variable region.

[0206] 1D6 humanization

[0207] The PDB structure and amino acid sequence corresponding to 1D6 VH is 1IHI, and the PDB structure and amino acid sequence corresponding to 1D6 VL is 5MYX. Using WinCoot, we substituted amino acid residues that differed from those in the 1D6 variable region. Each substitution was carefully evaluated for structural plausibility, and the results were all reasonable, with none affecting the model's secondary structure. GROMACS was used to calculate the lowest energy extremes of the structure, yielding a computer-simulated 3D structure of 1D6.

[0208] BioPhi (4) Software used for 1D6 variable region humanization:

[0209] Humanization setting: using Chothia (1) Amino acid numbering, using IMGT (2,3)Defining CDRs

[0210] Humanization method 1: Sapiens, humanization iteration 4 times, threshold greater than or equal to 10%.

[0211] Sapiens humanized 1D6 variable region:

[0212] >Antibody1 VH(Humanized Antibody 1D6 Sapiens 4iter parental Imgt CDRs BioPhi)

[0213] >Antibody1 VL(Humanized Antibody 1D6 Sapiens 4iter parental Imgt CDRs BioPhi)

[0214] Humanization method 2: Graft, the germline of both heavy and light chains are automatic, the threshold is greater than or equal to 10%, and the results are:

[0215] >Antibody1 VH(Humanized Antibody 1D6 Imgt Straight graft auto auto BioPhi)

[0216] >Antibody1 VL(Humanized Antibody 1D6 Imgt Straight graft auto auto BioPhi)

[0217] F11 humanization

[0218] The PDB structure and amino acid sequence corresponding to F11 VH is 6TKC, and the PDB structure and amino acid sequence corresponding to F11 VL is 5FB8. Using WinCoot, we substituted amino acid residues that differed from those in the F11 variable region. Each substitution was carefully evaluated for structural plausibility, and the results were all reasonable, with none affecting the model's secondary structure. GROMACS was used to calculate the lowest energy extremes of the structure, resulting in a computer-simulated 3D structure of F11.

[0219] BioPhi software was used for humanization of the F11 variable region

[0220] Humanization settings: use Chothia amino acid numbering and IMGT to define CDRs

[0221] Humanization method 1: Sapiens, humanization iteration 4 times, threshold greater than or equal to 10%

[0222] Sapiens humanized F11 variable region:

[0223] >Antibody1 VH(Humanized Antibody F11 Sapiens 4iter parental Imgt CDRs BioPhi)

[0224] >Antibody1 VL(Humanized Antibody1 Sapiens 4iter parental Imgt CDRs BioPhi)

[0225] Humanization method 2: Graft, the germline of both heavy and light chains are automatic, the threshold is greater than or equal to 10%, and the results are:

[0226] >Antibody1 VH(Humanized Antibody F11 Imgt Straight graft auto auto BioPhi)

[0227] >Antibody1 VL(Humanized Antibody1 Imgt Straight graft auto auto BioPhi)

[0228] When the humanized sequences of 1D6 and F11 were incorporated into the computer-simulated 3D structures of 1D6 and F11, no changes in the secondary structure were observed. Energy minima calculations revealed no significant changes in the secondary structure. The humanized 1D6 and F11 antibodies retained the binding capacity of their parental antibodies.

[0229] Example 4: Monoclonal Antibodies Inhibit Tumor Growth in Mice

[0230] This example studies the effect of antibody F11 on tumor proliferation in a subcutaneous transplanted tumor model of human malignant melanoma or human triple-negative breast cancer CB-17 SCID mice.

[0231] A-375 cells (malignant melanoma cell line) and HCC1937 cells (human breast cancer cells) were purchased from American Tissue Culture Collection (ATCC, Rockville, MD, USA).

[0232] CB-17 SCID mice, weighing 18 - 20 g, female, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK(Beijing)2016 - 0006), and were housed in a SPF - level environment. The indoor temperature was controlled at 23 ± 2 °C, and they had free access to food and water. The total number of animals was 32. They were adaptively housed for 3 days before inoculation.

[0233] A - 375 cells or HCC1937 cells in the logarithmic growth phase were taken to prepare a cell suspension with a concentration of 2.5×10 6 cells / ml. 0.2 ml per mouse was inoculated into the right axilla of nude mice to establish an A - 375 cell or HCC1937 mouse xenograft tumor model. On the day after tumor cell inoculation, each group of tumor - bearing mice was randomly divided into an experimental group (8 mice) and a control group (8 mice), marked, and then administered drugs.

[0234] Drug administration design: F11 or buffer was injected via the tail vein, for a total of 4 times. The first drug administration was on the day after tumor cell inoculation, which was the 1st day of drug administration, and then drugs were administered on the 3rd, 7th, and 13th days. Among them, the experimental group was given F11 (10 mg / Kg), and the blank control group was given buffer (equal volume to F11). See Table 5.

[0235] Table 5. Experimental design for mouse tumors

[0236] The diameter of the mouse xenograft tumor was measured with a vernier caliper. Using the method of measuring tumor diameter, the anti - tumor effect of the test substance was dynamically observed. The tumor diameter was measured once every 3 days, and the body weight of the mice was checked every 3 days. 60 days after tumor cell inoculation, all mice were sacrificed by the carbon dioxide method. After opening the abdomen for observation, the tumors were removed, and irrelevant tissues were carefully removed, washed 2 - 3 times with D - Hanks solution to wash away the blood, drained, and stored. The weight and volume were measured.

[0237] The calculation formula for tumor volume (TV) is:

[0238] Calculation of tumor volume measurement (mm 3 ) = 0.5[w1×(w2) 2 ​​​​​​The results of the A-375 cell transplantation experiment are shown in Figure 4. There was no significant difference in the body weight of the mice in the different groups. However, the tumor volume in the F11 group was significantly reduced compared to the blank control group, suggesting that the monoclonal antibody F11 can inhibit the growth of A-375 cell transplanted tumors. (Three control mice died within 60 days of tumor cell inoculation.)

[0241] The results of the HCC1937 cell transplantation experiment are shown in Figure 5. There was no significant difference in weight between the mice in the various groups; however, the tumor volume in the F11 group was significantly reduced compared to the blank control group, suggesting that the monoclonal antibody F11 can inhibit the growth of HCC1937 cell transplanted tumors. (Two control mice died within 60 days of tumor cell inoculation.)

[0242] Example 5. Preparation and characterization of fully human monoclonal antibodies

[0243] Fusion proteins GST-ZZS01 and GST-ZZS05 were used to immunize CAMouse (immune gene humanized mice of Chongqing Jinmai Biotechnology Co., Ltd., patent application publication number: CN 108486126 A). After routine immunization, the spleen and bone marrow of the mice were ground and resuspended. Density gradient centrifugation was performed to enrich monocytes. CD138+ B cells were isolated using a CD138+ kit. After plasma cells were isolated, human antibodies were obtained using the following two methods:

[0244] 1. Introduce the isolated plasma cells into the Beacon instrument (Manufacturer: BLI, Model: BSN0079). The positive clones obtained by BEACON for the target protein are then sequenced for the antibody gene.

[0245] 2. Mix the SP2 / 0 cell suspension and the spleen (plasma) cell suspension for cell fusion. After fusion, resuspend the fused cells in fusion medium, mix well to prepare a cell suspension, and then plate into a 96-well plate. Place the fused 96-well plate in a 5.5% CO2, 37°C CO2 incubator for 5-8 days. Transfer the supernatant from the cell culture plate to the corresponding ELISA test plate (coated with the immunizing antigen, test antigen, and counter-screening protein), add the hybridoma master clone supernatant (primary antibody), and incubate at 37°C for 60 minutes. Then, add the secondary antibody Anti-Human IgG (H&L) (GOAT) Antibody Peroxidase Conjugated (Min X Bv Ch Gt GP Ham Hs Ms Rb Rt & Sh Serum Proteins) and incubate at 37°C for 30 minutes. Then, add TMB colorimetric solution, incubate at 25°C for 15 minutes, and terminate the reaction with HCl. Cells from the screened positive wells are subcloned by limiting dilution. After subcloning, the 96-well plate was placed in a 37°C, 5%-8% CO2 incubator for 6-11 days. After screening for monoclonal antibody-expressing cells, the antibody gene was sequenced.

[0246] After combining the two methods to find the monoclonal antibody-expressing cells obtained, the NGS high-throughput sequencing method was used to measure the gene sequence of the antibody variable region to obtain the antibody variable region sequence.

[0247] Comparison of the detected antibody genes with the gene sequences in the IMGT database (2,3) , confirm that all antibody gene sequences are human antibody gene sequences. Recombinant human antibodies were expressed in CHO, and the expressed antibodies were tested for their corresponding antigen affinity using the Biacore method (see the previous example). (4) The CDR sequence was obtained using the software. The amino acid sequence of the antibody variable region was numbered using Chothia (1) Method, using IMGT (2,3) The method was defined to determine the CDR. The results are shown in Tables 6 and 7.

[0248] Table 6. List of human antibodies against zzs01

[0249] Table 7. List of human antibodies against zzs05

[0250] The corresponding heavy chain constant region and light chain constant region sequences are as follows:

[0251] Heavy chain constant region (SEQ ID NO: 417):

[0252] Light chain constant region (SEQ ID NO: 418):

[0253] Example 6: Human monoclonal antibody-mediated ADCC on tumor organoid cells

[0254] 6.1 Detection of the ADCC Effect of NK Cells on Pancreatic Cancer and Small Cell Lung Cancer Organoids Mediated by the Human Anti-ZZS05 Antibody h32 (also named A-1P13) (Beijing Daxiang Technology Co., Ltd.)

[0255] Experimental cells:

[0256] Organoids: DXPC-ZY-001 pancreatic cancer organoid (derived from a patient with stage III poorly differentiated pancreatic cancer), DXSCLC-ZY-001 small cell lung cancer organoid (derived from a patient with stage IV poorly differentiated small cell lung cancer)

[0257] NK cells: Commercially available PBMC-expanded NK cells DXNK-ZY-001

[0258] Experimental Antibodies:

[0259] Human anti-ZZS05 antibody A-1P13 (experimental group)

[0260] Humanized anti-influenza A protein HA antibody IgG1 (H7N9 (A / Anhui / 2013)) (control group)

[0261] Experiment Overview:

[0262] A co-culture model of tumor organoids and immune cells was established using a 384-well plate. Cultured tumor organoids were digested into single cells and stained for live cell tracking (Calcein, AM). Organoid tumor cells were then incubated with the test drug A-1P13 for 20 minutes. Natural killer (NK) cells were co-cultured with the tumor organoid cells at a 5:1 effector-target ratio. After a 5-hour incubation, dead cell tracking staining (SYTOX-Red) was performed and analyzed by flow cytometry.

[0263] Experimental groups:

[0264] Experimental results:

[0265] The results for pancreatic cancer organoids are shown in Figure 6A. Statistical analysis of apoptosis in tumor organoids in the ADCC model was performed. T test "*": P < 0.05, "**": P < 0.01. Compared with the organoid alone (O) group, organoid apoptosis in the O + NK + PBS / IgG group was significantly increased, with no significant differences between the O + NK + PBS and O + NK + IgG groups. Compared with O + NK + PBS and O + NK + IgG, apoptosis in organoids in the 10 μg / mL drug-treated group was most significantly increased. There were no significant differences between the drug-treated groups.

[0266] The results for small cell lung cancer organoids are shown in Figure 6B. Statistical analysis of apoptosis in tumor organoids in the ADCC model was performed. T test: *: P < 0.05, **: P < 0.01. Compared with the O+NK+PBS group, apoptosis in organoids treated with A-1P13 was significantly increased, while no significant differences were observed between the O+NK+PBS and O+NK+IgG groups. Compared with O+NK+PBS, apoptosis was most significantly increased in the 0.1 μg / mL A-1P13-treated group. No significant differences were observed between the drug-treated groups.

[0267] 6.2 Detecting the Effects of Humanized Anti-ZZS05 Antibody h35 (Also Named A-1P22) on Different Tumor Organoids (Shanghai Jiefang Kairui Biotechnology Co., Ltd.)

[0268] Experimental Design:

[0269] 1) Construction of organoids for colorectal cancer (CRC), lung cancer (LC), gastric cancer (GC), endometrial cancer (EC), breast cancer (BC), pancreatic cancer (PADC), liver cancer (HCC), and ovarian cancer (AOC);

[0270] 2) After organoid construction was completed, the cells were divided into the organoid + PBS group, the organoid + NK cell group, the organoid + NK cell + candidate antibody (h35) group, and the organoid + NK cell + tumor-unrelated antibody (rabbit anti-human PSA antibody) group;

[0271] 3) Co-culture of organoids with candidate antibodies and NK cells: Organoids were cultured in 96-well plates and divided into groups: organoid + PBS, organoid + NK cells, organoid + NK cells + candidate antibodies, and organoid + NK cells + tumor-unrelated antibodies. Live cell imaging was performed, with continuous brightfield images of organoid growth. After 48 hours of continuous drug administration, live-death staining was performed and images were taken. Organoid viability and LDH release were also measured.

[0272] 4) Three wells in each group were biological replicates.

[0273] Test method:

[0274] Primary organoid culture

[0275] (1) After sampling, the cancer tissue must be washed at 2-8°C. Prepare several culture dishes and add 4°C pre-cooled primary culture buffer (with double antibody and gentamicin).

[0276] (2) Place the tissue in a culture dish and wash it three times with primary culture buffer B to remove impurities. Use ophthalmic scissors or a scalpel to gently press the cancerous tissue to remove some red blood cells and cut it into pieces with a volume of about 1-3 mm. 3 organizational blocks.

[0277] (3) Digest the tissue with primary tissue digestion solution C at 37°C with shaking for 25-30 min (observe the digestion status at any time during the digestion process).

[0278] (4) Take a small amount of liquid and observe it under a microscope. If a large number of crypts are observed under the microscope, add three times the volume of primary culture buffer to terminate the digestion.

[0279] (5) Filter through a 100 μm pore size mesh, collect the filtrate, centrifuge at 300 g for 5 min, remove the supernatant, add primary culture buffer B, and resuspend and centrifuge.

[0280] (6) Matrigel calculation: After step 6, observe the volume of the collected tissue and add 40 times the volume of the tissue to the Matrigel to resuspend and plate.

[0281] (7) For example, a 24-well cell culture plate was plated with 25 μl of tissue matrix gel mixture per well (operated at 4°C).

[0282] (8) Place the prepared culture plate in a 37°C incubator for 10-15 minutes to gel, add intestinal cancer organoid culture medium A (restore to room temperature) and culture.

[0283] Organoid subculture

[0284] (1) Remove the culture medium with a pipette and add 1-2 ml of 4°C organoid subculture buffer G to each well and incubate for 2 minutes.

[0285] (2) Gently pipette the Matrigel into a 15 ml centrifuge tube and incubate at 4°C for 10 min. (6-8 wells per group) Centrifuge at 300 g for 5 min, discard the supernatant, add 2 ml of Organoid Subculture Digestion Buffer D, and digest for 3 min. Add Organoid Subculture Buffer G to terminate digestion, centrifuge at 300 g for 5 min, discard the liquid, and proceed to step 4.

[0286] (3) After organoids are collected, add Matrigel to resuspend them, and 30 μl of Matrigel mixture is plated in each well of a 24-well cell culture plate. Place the plate in an incubator for 10-15 minutes and then add 500 μl of organoid culture medium A.

[0287] Co-culture of organoids with candidate antibodies and NK cells

[0288] (1) Organoids were incubated with organoid culture medium containing 200 ng / mL IFNγ for 24 h.

[0289] (2) Remove the culture medium with a pipette and add 1-2 ml of 4°C organoid subculture buffer G to each well and incubate for 2 minutes.

[0290] (3) Gently pipette the matrix gel, collect it in a 15 ml centrifuge tube, and let it stand at 4°C for 10 minutes (each group consists of 6-8 wells).

[0291] (4) After organoid collection, resuspend the organoids in NK cell culture medium and add 50ul of organoid mixture to a 96-well cell culture plate (the optimal number of organoids is 10-20 per well). Note: Organoid blank group resuspended organoids in PBS.

[0292] (5) The collected NK cells were co-incubated with the candidate antibody (5 μg / ml) and the tumor-independent antibody (5 μg / ml), and the NK cell culture medium was replenished to 100 μl. The ratio of NK cells to organoids was 500:1.

[0293] (6) High-content continuous photography for 48 hours to organize the results.

[0294] (7) Perform cell viability staining according to the kit instructions and take photos.

[0295] (8) Detection of LDH (lactate dehydrogenase) release.

[0296] Experimental results:

[0297] Results of continuous video of organoids, candidate antibodies, and NK cells:

[0298] Compared with the organoid + PBS group and the organoid + NK group, the organoid + NK + candidate antibody group had increased NK cell recognition in the following groups after 48 hours of action: AOC: ovarian cancer organoids; BC breast cancer organoids; CRC colorectal cancer organoids; EC: endometrial cancer organoids; GC gastric cancer organoids; HCC liver cancer organoids; LC lung cancer organoids; PADC: pancreatic cancer organoids.

[0299] Results of fluorescent live-dead staining of organoids, candidate antibodies, and NK cells (Figure 7):

[0300] Compared with the organoid + PBS group and the organoid + NK group, the organoid + NK + candidate antibody group had increased cell apoptosis in the following groups after 48 hours of action: AOC: ovarian cancer organoids; BC: breast cancer organoids; EC: endometrial cancer organoids; HCC: liver cancer organoids; LC: lung cancer organoids; PADC: pancreatic cancer organoids.

[0301] LDH release results (Figure 8):

[0302] EC: Endometrial cancer organoids

[0303] Compared with the organoid + PBS group and the organoid + NK group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action, while there was no significant change in the organoid + NK + tumor-related antibody group; compared with the organoid + NK group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action.

[0304] CRC: Colorectal cancer organoids

[0305] Compared with the organoid + PBS group and the organoid + NK group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action, while there was no significant change in the organoid + NK + tumor-related antibody group; compared with the organoid + NK group, there was no significant change in the LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0306] BC: Breast cancer organoids

[0307] Compared with the organoid + PBS group and the organoid + NK group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action, while there was no significant change in the organoid + NK + tumor-related antibody group; compared with the organoid + NK group, there was no significant change in the LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0308] GC: Gastric cancer organoids

[0309] Compared with the organoid + PBS group, the organoid + NK group, the organoid + NK + candidate antibody group, and the organoid + NK + tumor-related antibody group, there was no significant change in LDH release after 48 hours of action; compared with the organoid + NK group, there was no significant change in LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0310] AOC: Ovarian Cancer Organoids

[0311] Compared with the organoid + PBS group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action, while there was no significant change in the organoid + NK group and organoid + NK + tumor-related antibody group; compared with the organoid + NK group, there was no significant change in the LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0312] PADC: Pancreatic Cancer Organoids

[0313] Compared with the organoid + PBS group, the LDH release in the organoid + NK group and the organoid + NK + candidate antibody group increased significantly after 48 hours of action, while there was no significant change in the organoid + NK + tumor-related antibody group; compared with the organoid + NK group, there was no significant change in the LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0314] HCC: Liver cancer organoids

[0315] Compared with the organoid + PBS group, the LDH release in the organoid + NK group, organoid + NK + candidate antibody group, and organoid + NK + tumor-unrelated antibody group increased significantly after 48 hours of action; compared with the organoid + NK group, there was no significant change in the LDH release in the organoid + NK + candidate antibody group after 48 hours of action.

[0316] LC: lung cancer organoids

[0317] Compared with the organoid + PBS group, the LDH release in the organoid + NK group, organoid + NK + candidate antibody group, and organoid + NK + tumor-related antibody group increased significantly after 48 hours of action; compared with the organoid + NK group, the LDH release in the organoid + NK + candidate antibody group increased significantly after 48 hours of action.

[0318] References:

[0319] 1.Chothia C,Lesk a M.Canonical structures for the hypervariable regions of immunoglobulins.J Mol Biol.(1987)196:901–17.doi:10.1016 / 0022-2836(87)90412-8

[0320] 2.Lefranc,M.P.IMGT,the international ImMunoGeneTics information system:A standardized approach for immunogenetics and immunoinformatics.Immunome Res 2005,1,3.

[0321] 3.Lefranc,M.P.;Giudicelli,V.;Kaas,Q.;Duprat,E.;Jabado-Michaloud,J.;Scaviner,D.;Ginestoux,C.;Clement,O.;Chaume,D.;Lefranc,G.IMGT,the international ImMunoGeneTics information system.Nucleic Acids Res.2005,33,D593–D597.

[0322] 4.David Prihoda,Jad Maamaryc,Andrew Waightd,Veronica Juand,Laurence Fayadat-Dilmand,Daniel Svozil a,e,and Danny A.Bitton:BioPhi:A platform for antibody design,humanization,and humanness evaluation based on natural antibody repertoires and deep learning.

[0323] 5.Collaborative Computational Project No.4(CCP4)-Software for Macromolecular X-Ray Crystallography.

[0324] 6.A free and open-source software suite for high-performance molecular dynamics and output analysis。

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody i) specifically binds to the extracellular region of the cTAGE5 protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or ii) specifically binds to an immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a polyclonal antibody, or the antibody is a monoclonal antibody.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and comprises a heavy chain CDR and a light chain CDR selected from any one of the following groups:

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise an amino acid sequence selected from any one of the following groups or respectively comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity with an amino acid sequence selected from any one of the following groups:

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a humanized antibody.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the humanized antibody i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24; ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; or iv) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

30.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody comprises the heavy chain shown in SEQ ID NO: 3, and the light chain shown in SEQ ID NO: 4; or the antibody comprises the heavy chain shown in SEQ ID NO: 13, and the light chain shown in SEQ ID NO:

14.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof binds to the extracellular region of the cTAGE5 protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2 or the immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2. D Value less than 1×10 -7 M, preferably less than 1×10 -8 M, more preferably less than 1×10 -9 M, more preferably less than 1×10 -10 M.

10. An antibody conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a therapeutic moiety selected from a cytotoxin, a radioactive isotope or a biologically active protein conjugated to the antibody or antigen-binding fragment thereof.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the antibody conjugate according to claim 10, and a pharmaceutically acceptable carrier.

12. A method for treating and / or preventing a tumor in a patient, the method comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody conjugate according to claim 10, or the pharmaceutical composition according to claim 11.

13. The method of claim 12, wherein the tumor is a cTAGE5 protein-associated tumor, such as a tumor that overexpresses cTAGE5 protein, wherein the cTAGE5 protein has an extracellular region, and the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

14. The method of claim 12 or 13, wherein the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung cancer, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer.

15. The method of any one of claims 12-14, further comprising administering to the patient other anti-tumor treatments, such as administration of chemotherapeutic agents, antibodies targeting other tumor-specific antigens, or radiation therapy.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody conjugate according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating and / or preventing tumors.

17. The use according to claim 16, wherein the tumor is a cTAGE5 protein-related tumor, such as a tumor overexpressing cTAGE5 protein, wherein the cTAGE5 protein has an extracellular region, and the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

18. The method of claim 17, wherein the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung cancer, glioma, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, endometrial cancer, colorectal cancer.

19. A method for detecting the presence of cTAGE5 protein or the expression level of cTAGE5 protein in a biological sample, comprising: contacting the biological sample and an optional control sample with the antibody of the present invention, such as a monoclonal antibody or an antigen-binding fragment thereof, under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 and the cTAGE5 protein; and Detection of complex formation, wherein the formation of the complex in the biological sample or the difference in complex formation between the biological sample and a control sample indicates the presence of cTAGE5 protein in the sample or the expression level of cTAGE5 protein, The cTAGE5 protein has an extracellular region comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

20. The method of claim 19, wherein the biological sample is an ex vivo sample, such as a tissue sample, a blood sample, a lymph sample, etc.

21. The method of claim 19 or 20, which is an immunohistochemical assay or an immunofluorescence assay.

22. A method for detecting the presence of a tumor in a patient, comprising detecting, for example by the method of any one of claims 19 to 21, the expression level of cTAGE5 protein in a biological sample from the patient, wherein the presence of cTAGE5 in the biological sample of the patient or the expression level of cTAGE5 in the biological sample of the patient is higher than the expression level of cTAGE5 from a control biological sample indicates the presence of a tumor in the patient, The cTAGE5 protein has an extracellular region comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

23. The method of claim 22, wherein the tumor is a cTAGE5 protein-associated tumor, such as a tumor overexpressing cTAGE5 protein, wherein the cTAGE5 protein has an extracellular region, and the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

24. The method of claim 23, wherein the tumor is selected from nasopharyngeal carcinoma, malignant melanoma, lung cancer, glioma, skin cancer, glossopharyngeal carcinoma, lymphoma (including B-cell lymphoma or T-cell lymphoma), liver cancer, cervical cancer, thyroid cancer, colon cancer, ovarian cancer, brain tumor, prostate cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, endometrial cancer, colorectal cancer.

25. An isolated immunogenic polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, or an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:1 or SEQ ID NO:

2.

26. Use of the isolated immunogenic polypeptide of claim 25 in the preparation of antibodies.

27. A method for preparing antibodies, the method comprising immunizing an animal with the isolated immunogenic polypeptide of claim 25, and then isolating antibodies that specifically bind to the polypeptide from the animal or obtaining sequence information of antibodies that specifically bind to the polypeptide.

28. A pharmaceutical composition comprising the isolated immunogenic polypeptide of claim 25 and a pharmaceutically acceptable carrier.

29. Use of the isolated immunogenic polypeptide of claim 25 in the preparation of a medicament, for example, the medicament is used for treating and / or preventing tumors.

30. A method for preventing and / or treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the isolated immunogenic polypeptide of claim 25 or the pharmaceutical composition of claim 28.

31. A method for detecting the presence and / or amount of antibodies that specifically bind to cTAGE5 protein in a biological sample, comprising 1) contacting the isolated immunogenic polypeptide of claim 25 with the biological sample, and 2) detecting the presence and / or amount of antibodies that bind to the isolated immunogenic polypeptide.

32. Use of the isolated immunogenic polypeptide of claim 25 in the preparation of a kit for detecting the presence and / or amount of antibodies that specifically bind to a cTAGE5 protein in a biological sample.

33. Use of a cTAGE5 protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or comprising an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or comprising an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, preferably comprising the amino acid sequence shown in SEQ ID NO: 2 as a tumor marker.

34. A tumor screening method, the method comprising detecting the presence or amount of cTAGE5 protein in a biological sample from a subject, the cTAGE5 protein comprising the amino acid sequence shown in SEQ ID NO:1 or 2, or comprising an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:1, or comprising an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO:1, preferably comprising the amino acid sequence shown in SEQ ID NO:

2.

35. A tumor screening method, the method comprising detecting the presence and / or amount of an antibody that specifically binds to a cTAGE5 protein in a biological sample from a subject, wherein the antibody specifically binds to an extracellular region of the cTAGE5 protein, wherein the extracellular region of the cTAGE5 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or comprises an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1, or comprises an amino acid sequence having one or more (such as 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions relative to SEQ ID NO: 1, preferably comprises the amino acid sequence shown in SEQ ID NO: 2.