ANTICLAUDIN ANTIBODIES 18.2 AND USES OF THE SAME

专利摘要:
the present invention relates to anti-cldn18.2 antibodies and antigen-binding fragments thereof. nucleic acids encoding antibodies are also described, compositions comprising antibodies and methods of producing antibodies and use of antibodies for treating or preventing diseases such as cancer and / or inflammatory disease.
公开号:BR112020015479A2
申请号:R112020015479-2
申请日:2019-03-06
公开日:2020-12-08
发明作者:Minghan Wang；Hui Zou；Haiqun Jia
申请人:Phanes Therapeutics, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims priority for provisional US patent application No. 62 / 640,288, filed on March 8, 2018; and for provisional US patent application No. 62 / 714,254, filed on August 3, 2018. Each disclosure is incorporated herein in its entirety, for reference. FIELD OF THE INVENTION
[0002] [0002] This invention relates to monoclonal anti-viral antibodies 18.2 (CLDN18.2), nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors and compositions comprising the antibodies. Methods are also provided to produce the antibodies and methods for using the antibodies to treat diseases such as cancer and inflammatory diseases and / or associated complications. REFERENCE TO THE SEQUENCE LISTING SENT ELECTRONICALLY CAMENTE
[0003] [0003] This order contains a sequence listing, which is sent electronically via EFS-Web as an ASCII formatted sequence listing with a file name "689204.9W0 Sequence Listing" and a creation date of February 26, 2019 and with a size of 74 kb. The list of strings sent via EFS-Web is part of the specification and is incorporated by reference in its entirety. BACKGROUND OF THE INVENTION
[0004] [0004] Claudine 18.2 (CLDN18.2), also known as claudidine-18a2.1, belongs to the transmembrane proteins of the claudine family (CLDN) of at least 27 isoforms in humans. Claudins are the main structural components of the close junction between epithelial cells and function as ionic pores to regulate the paracellular permeability of cations and anions (Sahin et al., Physiol Rev. 2013; 93: 525-569). CLDN18 expression is usually limited to lung and stomach tissues. CLDN18 has two splice variants. CLDN18.1 is the specific variant for the lung, while CLDN18.2 is the specific variant for the stomach. The splice variants differ in their 69 N-terminal amino acid residues due to the alternative splice of the first exon (Niimi et al., Mol Cell Biol. 2001; 21: 7380-7390). Studies with mice with CLDN18.2 deactivated genes suggest that CLDN18.2 plays a critical role in preventing gastric acid leakage in the stomach lumen (Hayash et al., Gastroenterology 2012; 142: 292-304).
[0005] [0005] The unregulated expression of claudins is detected in many cancers and can contribute to tumorigenesis and cancer invasion (Singh et al, J Oncology 2010; 2010: 541957). CLDN18.2 expression is elevated in pancreatic ductal adenocarcinomas (PDAC) (Tanaka et al., J Histochem Cytochem. 2011; 59: 942-952), esophageal tumors, non-small cell lung cancers (NSCLC), cancers ovarian (Sahin et al., Hu Cancer Biol. 2008; 14: 7624-7634), bile duct adenocarcinomas (Keira et al., Virchows Arch. 2015; 466: 265-277) and cholangiocarcinomas (Shinozaki et al., Vir - chows Arch. 2011; 459: 73-80). CLDN18.2 is an ideal target for precision-oriented anti-cancer biologics for positive CLDN18.2 tumors, since CLDN18.2 is not expressed in any normal tissue other than differentiated gastric epithelial cells, unattainable by compounds of large molecules administered intravenously. BRIEF SUMMARY OF THE INVENTION
[0006] [0006] In a general aspect, the invention relates to isolated monoclonal antibodies or antigen-binding fragments that specifically bind to claudin 18.2 (CLDN18.2).
[0007] [0007] Isolated monoclonal antibodies or antigen-binding fragments thereof are provided comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1) , LCDR2 and LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 33, 34, 35, 63, 64 and 65, respectively; (2) SEQ ID NOs: 21, 22, 23, 51, 52 and 53, respectively; (3) SEQ ID NOs: 24, 25, 26, 54, 55 and 56, respectively; (4) SEQ ID NOs: 27, 28, 29, 57, 58 and 59, respectively; (5) SEQ ID NOs: 30, 31, 32, 60, 61 and 62, respectively; (6) SEQ ID NOs: 36, 37, 38, 66, 67 and 68, respectively; (7) SEQ ID NOs: 39, 40, 41, 69, 70 and 71, respectively; (8) SEQ ID NOs: 42, 43, 44, 72, 73 and 74, respectively; (9) SEQ ID NOs: 45, 46, 47, 75, 76 and 77, respectively; or (10) SEQ ID NOs: 48, 49, 50, 78, 79 and 80, respectively; wherein the antibody or antigen binding fragment thereof specifically binds to CLDN18.2, preferably to human CLDN18.2.
[0008] [0008] Isolated monoclonal antibodies or antigen-binding fragments thereof are provided comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1) , LCDR2 and LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 93, 94, 95, 123, 124 and 125, respectively; (2) SEQ ID NOs: 81, 82, 83, 111, 112 and 113, respectively; (3) SEQ ID NOs: 84, 85, 86, 114, 115 and 116, respectively; (4) SEQ ID NOs: 87, 88, 89, 117, 118 and 119, respectively;
[0009] [0009] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9, 1.3, 5.7, 11, 13.15, 17 or 19, or a light chain variable region with a polypeptide sequence of at least 95%, at least 96%, at least 97% to SEQ ID NO: 10, 2, 4, 6, 8, 12, 14, 16, 18 or 20.
[0010] [0010] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a variable chain region lightweight having the polypeptide sequence of SEQ ID NO: 10; (b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4; (d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 6; (e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8; (f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14; (h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (i) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; (]) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20;
[0011] [0011] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment of it binds to CLDN18.2 and is capable of inducing effector-mediated tumor cell lysis through antibody-dependent cell cytotoxicity (ADCC), antibody-dependent phagocytosis (ADPC) and / or complement-dependent cytotoxicity (CDC), and / or mediate the recruitment of conjugated drugs, and / or form a bispecific antibody with another monoclonal antibody or antigen-binding fragment of the even with the effect of killing cancer.
[0012] [0012] In certain embodiments, the isolated monoclonal antibody or the antigen-binding fragment thereof is chimeric.
[0013] [0013] In certain embodiments, the isolated monoclonal antibody or the antigen-binding fragment of the same is human or humanized.
[0014] [0014] In certain embodiments, the humanized monoclonal antibody or the antigen-binding fragment thereof comprises: a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; B. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; ç. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; and. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148; f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 149; g. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150; H. a heavy chain variable region having the sequence
[0015] [0015] Isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments of the invention are also provided.
[0016] [0016] Vectors are also provided that comprise the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments of the invention.
[0017] [0017] Host cells are also provided which comprise the vectors comprising the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments of the invention.
[0018] [0018] In certain embodiments, a pharmaceutical composition is provided comprising an isolated monoclonal antibody or an antigen-binding fragment of the invention and a pharmaceutically acceptable carrier.
[0019] [0019] Methods are also provided to specifically target CLDN18.2, but not CLDN18.1, on a cancer cell surface in an individual who needs it, comprising administering to the individual a pharmaceutical composition of the invention - dog.
[0020] [0020] Cancer treatment methods are also provided to an individual who needs it, comprising administering to the individual the pharmaceutical compositions of the invention. Cancer can be any liquid or solid cancer, for example, it can be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, carcinoma hepatocellular, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL ), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.
[0021] [0021] Methods are also provided to treat an inflammatory disease in an individual who needs it, comprising administering to the individual a pharmaceutical composition of the invention.
[0022] [0022] Methods are also provided to produce a monoclonal antibody or antigen-binding fragment of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or the antigen-binding fragment thereof, in conditions to produce the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cell or culture.
[0023] [0023] Methods are also provided for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof, comprising combining the monoclonal antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier for obtaining the pharmaceutical composition.
[0024] [0024] Methods are also provided to determine the level of CLDN18.2 in an individual. The methods include (a) obtaining a sample from the individual; (b) placing the sample in contact with an antibody or antigen-binding fragment thereof of the invention; and (c) determining the level of CLDN18.2 in the individual. In certain modes, the sample is a tissue sample. The tissue sample can, for example, be a cancer tissue sample. In certain embodiments, the sample is a blood sample. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] [0025] The aforementioned summary, as well as the detailed description below of the preferred modalities of this application, will be better understood when read in conjunction with the attached drawings. However, it must be understood that the application is not limited to the precise modalities shown in the drawings.
[0026] [0026] Figs. 1A4-1D show the dose-dependent binding of purified chimeric anti-CLDN18.2 mAbs (VH and VL regions of mouse mAbs fused to the constant regions of the human IgG1 heavy chain and the kappa light chain, respectively) to a pool of cells HEK293 transfected stably with full-length human CLDN18.2 by FACS analysis.
[0027] [0027] Figs. 2A-2E show the selective binding of anti-CLDN18.2 chimeric mAbs to CLDN18.2 compared to CLDN18.1. They were used in the experiment of pools of HEK293 cells transfected with full-length human CLDN18.2 and CLDN18.1, respectively. The shaded gray peak is representative of the treatment group incubated with only the secondary antibody and the open black peak is representative of the treatment group incubated with both the primary chimeric mAb and the secondary antibody.
[0028] [0028] Figs. 3A-3C show the results of dose-dependent binding of chimeric anti-CLDN18.2 mAbs to a stable HEK293-CLDN18.2 cell line by FACS analysis.
[0029] [0029] Figs. 4A-4J show the histograms for binding the chimeric anti-CLDN18.2 mAbs to a stable HEK293-CLDN18.2 cell line. The shaded gray peak is representative of the treatment group incubated with only the secondary antibody and the open black peak is representative of the treatment group incubated with both primary chimeric mAb and the secondary antibody.
[0030] [0030] Figs. 5A-5G show the results of dose-dependent binding of humanized anti-CLDN18.2 mAbs to stable pools of HEK293-CLDN18.2 by FACS analysis.
[0031] [0031] Figs. 6A-GBE show the results of dose-dependent binding of humanized anti-CLDN18.2 mAbs to a stable HEK293-CLDN18.2 cell line by FACS analysis.
[0032] [0032] Fig. 7 shows the binding of humanized anti-CLDN18.2 mAbs to the NUGC-4 cancer cell line at the mAb concentration of 685 nM by FACS analysis.
[0033] [0033] Figs. 8A-8B show the results of antibody-dependent cell cytotoxicity (ADCC) activity of five humanized anti-CLDN18.2 mAbs and a chimeric anti-CLDN18.2 mAb. DETAILED DESCRIPTION OF THE INVENTION
[0034] [0034] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is hereby incorporated by reference, in its entirety. The discussion of documents, acts, materials, devices, articles or similars that were included in this specification is intended to provide context for the invention. Such a discussion is not an admission that any or all of these matters are part of the prior art with respect to any inventions revealed or claimed.
[0035] [0035] Unless otherwise defined, all technical and scientific terms used here will have the same meaning as is normally understood by the person skilled in the art to which the present invention belongs. Otherwise, certain terms used in this document have the meanings defined in the specification.
[0036] [0036] It should be noted that, As used here and in the appended claims, the singular forms “um”, “uma”, “o” and “a” include references in the plural, unless the context determines clearly the opposite.
[0037] [0037] Except where otherwise specified, any numerical values, such as a concentration or concentration range described herein, should be understood to be modified in all cases by the term "about". Therefore, a numerical value usually includes + 10% of the value recited. For example, a concentration of 1 mg / ml includes 0.9 mg / ml to 1.1 mg / ml. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used here, the use of a numeric range expressly includes all possible sub-ranges, all individual numeric values within that range, including an integer, within such ranges and fractions of values unless the context clearly indicates otherwise.
[0038] [0038] Unless otherwise stated, the term "at least" that precedes a series of elements must be understood to refer to all elements of the series. Those skilled in the art will recognize or be able to use with determination no more than the experimentation routine, many equivalents of the specific modalities of the invention described here. Such equivalents are intended to be encompassed by the invention.
[0039] [0039] As used herein, the terms "comprises", "understanding", "includes", "including", "has", "having", "contains" or "containing", or any other variation thereof, shall be understood to imply the inclusion of an integer or group of integers, but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open. For example, a composition, mixture, process, method, article or device comprising a list of elements is not necessarily limited to just those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article or apparatus. In addition, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present) ), and A and B are true (or present).
[0040] [0040] As used here, the conjunctive term "and / or" among various recited elements is understood to encompass both individual and combined options. For example, where two elements are joined by "and / or", a first option refers to the applicability of the first element without the second. A second option concerns the applicability of the second element without the first. A third option concerns the applicability of the first and second elements together. Any of these options is understood to fit the meaning and therefore meets the requirement of the term "and / or", as used here. The simultaneous applicability of more than one of the options is also understood as falling within the meaning and, therefore, satisfies the requirement of the term "and / or".
[0041] [0041] As used here, the term "consists of" or variations, such as "consist of" or "consisting of", as used throughout the specification and claims, indicate the inclusion of any integer or group of integers mentioned, but no whole numbers or additional groups of whole numbers can be added to the method,
[0042] [0042] As used here, the term "consists essentially of", and variations such as "consist essentially of" or "consisting essentially of", as used throughout the specification and claims, indicate the inclusion of any integer or group of integers mentioned, and the optional inclusion of any integer or group of integers that does not substantially alter the basic or new properties of the specified method or composition. See M.P.E.P. $ 2111.03.
[0043] [0043] As used herein, "individual" means any animal, preferably a mammal, more preferably a human being. The term "mammal", as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably a human.
[0044] [0044] The words "right", "left", "lower" and "upper" designate directions in the drawings to which reference is made.
[0045] [0045] It should also be understood that the terms "about", "approximately", "generally", "substantially" and similar terms used here when referring to a dimension or feature of a component of the preferred invention , indicates that the dimension / characteristic described is not a strict limit or parameter and does not exclude minor variations that are functionally the same or similar, as would be understood by one skilled in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the technique (for example, rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.
[0046] [0046] The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences (for example, anti-CLDN18.2 antibodies and polynucleotides that encode them, CLDN18.2 polypeptides and polynucleotides CLDN18.2 that make it difficult), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides equal, when compared and aligned for maximum matching, as measured using one of the following sequential comparison algorithms or by visual inspection.
[0047] [0047] For sequence comparison, normally a sequence acts as a reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the parameters of the sequence algorithm program are designated. The sequence comparison algorithm calculates the percentage of the sequence identity for the test sequences in relation to the reference sequence, based on the parameters of the designated program.
[0048] [0048] The ideal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by Needleman and Wunsch's homology alignment algorithm, J. Mol. Biol. 48: 443 (1970), for the search for the similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), for computer implementations using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see, in general, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing
[0049] [0049] Examples of suitable algorithms to determine the sequence identity percentage and the sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. The software to perform BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query string, which match or satisfy a positive value threshold score T when aligned with a word of the same length in a string database. T is called the neighborhood word score limit (Altschul et al, supra). These initial occurrences of neighborhood words act as seeds to initiate searches to find longer HSPs that contain them. The occurrences of words are then extended in both directions along each sequence until the cumulative alignment score can be increased.
[0050] [0050] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for residues with mismatch; always < O). For amino acid sequences, a score matrix is used to calculate the cumulative score. The extent of word occurrences in each direction is interrupted when: the cumulative alignment score falls by the amount X of its maximum value reached; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments;
[0051] [0051] In addition to calculating the percent identity of the sequence, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat '. Acad. Sci. USA 90: 5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the probability of a minor sum in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01 and with the maximum preference less than about 0.001.
[0052] [0052] Another indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under strict conditions. Antibodies.
[0053] [0053] The invention generally refers to isolated anti-CLDN18.2 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors and compositions comprising the antibodies. Methods for producing antibodies and methods for using antibodies to treat diseases such as cancer and inflammatory diseases. The antibodies of the invention have one or more desirable functional properties, including, but not limited to, high affinity binding to CLDN18.2, high specificity to CLDN18.2, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody dependent phagocytosis (ADPC) and / or antibody dependent mediated cell cytotoxicity (ADCC) against cells expressing CLDN18.2, and the ability to inhibit tumor growth in individuals and animal models when administered alone or in combination with others anticancer therapies.
[0054] [0054] In a general aspect, the invention relates to isolated monoclonal antibodies or antigen-binding fragments that bind to CLDN18.2.
[0055] [0055] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including human, humanized, compound and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that have specific binding to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five main classes (IgA, IgD, IgE, IgG and IgM) depending on the sequence of heavy chain constant domain amino acids. IgA and IgG are further subclassified as IgA1, IgA2, IgG1, IgG2, IgG3 and I9gG4 isotypes. Thus, the antibodies of the invention can be of any of the five main classes or corresponding subclasses. Preferably, the antibodies of the invention are IgG1, IgG2, IgG3 or I1g9G4. The antibody light chains of vertebrate species can be attributed to one of two clearly distinct types, kappa and lambda, based on the amino acid sequences of their constant domains. Consequently, the antibodies of the invention can contain a kappa or lambda light chain constant domain. According to specific modalities, the antibodies of the invention include constant regions of heavy and / or light chain of human or rat antibodies. In addition to the heavy and light constant domains, the antibodies contain an antigen binding region that consists of a variable light chain region and a variable heavy chain region, each of which contains three domains (i.e., determining regions complementarity 1-3; CDR1, CDR2 and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR 2 and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2 and HCDR3.
[0056] [0056] As used herein, the term "isolated antibody" refers to an antibody substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to CLDN18.2 is substantially free of anti - bodies that do not bind to CLDN18.2). In addition, an isolated antibody is substantially free of other cellular materials and / or chemicals.
[0057] [0057] As used here, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that comprise the population are identical, except for possible naturally occurring mutations that can be present in small amounts.The monoclonal antibodies of the invention can be made by the hybridoma method, phage display technology, simple lymphocyte gene cloning technology or by DNA methods For example, monoclonal antibodies can be produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, such as a transgenic mouse or rat, with a genome comprising a human heavy chain transgene and a human transgene. light chain.
[0058] [0058] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, a Fab, a Fab ', an F (ab') 2, a fragment Fv, a stabilized disulfide Fv fragment (dsFv), one (dsFv) 2, a bispecific dsFv (dsFv- dsFv '), a disulfide stabilized diabody (diabody ds), a single chain antibody molecule ( scFv), a single domain molecule (sdab), a scFv dimer (bivalent diabody), a multi-specific antibody formed from a part of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a divalent domain antibody, or any other antibody fragment that binds to an antigen, but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the original antibody or an original antibody fragment binds. According to specific embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region and a heavy chain Fd segment. According to other specific embodiments, the antigen-binding fragment comprises Fab and F (ab ').
[0059] [0059] As used herein, the term "single chain antibody" refers to a conventional single chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the field, comprising a heavy chain variable region and a heavy chain constant region or comprising only a variable region of heavy chain. heavy chain.
[0060] [0060] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technique known in the field. This definition of a human antibody includes intact or full-length antibodies, fragments thereof and / or antibodies comprising at least one human heavy and / or light chain polypeptide.
[0061] [0061] As used herein, the term "humanized antibody" refers to a non-human antibody modified to increase the sequence homology to that of a human antibody, so that the antibody's antigen binding properties are maintained, but their antigenicity in the human body is reduced.
[0062] [0062] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of the light and heavy chains often corresponds to the variable region of an antibody derived from a mammalian species (for example, mouse, rat, rabbit, etc.) with the specificity, affinity and desired capacity, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (eg, human) to avoid obtaining an immune response in that species.
[0063] [0063] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first plurality immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality have binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In a fashion, the first and second epitopes overlap or substantially overlap. In one modality, the first and second epitopes do not overlap or do not substantially overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, a multi-specific antibody comprises a third, fourth or fifth variable domain of immunoglobulin. In one embodiment, a multispecific antibody is a bispecific antibody molecule, a triespecific antibody molecule, or a tetra-specific antibody molecule.
[0064] [0064] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In a fashion, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In a modality, a bispecific antibody comprises a variable domain sequence heavy chain and a light chain variable domain sequence that has binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that has binding specificity for one second epitope. In one embodiment, a bispecific antibody comprises an antibody medium, or fragment thereof, having binding specificity for a first epitope and anti-body medium, or fragment thereof, having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope. In a modality, the first epitope is located in CLDN18.2 and the second epitope is located in PD-1, PD-L1, TIM-3, LAG-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD3, CD73, CDA47, TIP-1, apelin, DLL3, alpha folate receptor or other immune suppressors associated with the tumor or surface antigens.
[0065] [0065] As used here, the term "CLDN18.2" refers to claudin 18 variant 2, claudin-18.2 or claudin-18a2.1, which belongs to the claudin family of transmembrane proteins. CLDN18.2 is specifically expressed on the surface of epithelial cells in the stomach (Niimi et al., Mol Cell Biol. 2001; 21: 7380-7390) and becomes one of the main structural components of the close junction between epithelial cells (Sahin et al., Physiol Rev. 2013; 93: 525-569). The term "human CLDN18.2" refers to a CLDN18.2 originating from a human being. A sequence
[0066] [0066] As used herein, an antibody that "specifically binds to CLDN18.2" refers to an antibody that binds to a CLDN18.2, preferably a human CLDN18.2, with a KD of 1x107 M or less - knots, preferably 1x10 M or less, preferably 5x 10º M or less, 1x10º M or less, 5x107 * º M or less, or 1x10º M or less. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (ie, Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined using surface plasmon resonance, such as using a biosensor system, for example, a Biacore & system, or using bio-layer interferometry technology, such as an Octet system RED96.
[0067] [0067] The lower the KD value of an antibody, the greater the affinity that the antibody binds to a target antigen.
[0068] [0068] According to a specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), an HCDR2, an HCDR3, a light chain complementarity determining region 1 (LCDR1), an LCDR 2 and an LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 33, 34, 35, 63, 64 and 65, respectively; (2) SEQ ID NOs: 21, 22, 23, 51, 52 and 53, respectively; (3) SEQ ID NOs: 24, 25, 26, 54, 55 and 56, respectively; (4) SEQ ID NOs: 27, 28, 29, 57, 58 and 59, respectively; (5) SEQ ID NOs: 30, 31, 32, 60, 61 and 62, respectively; (6) SEQ ID NOs: 36, 37, 38, 66, 67 and 68, respectively;
[0069] [0069] According to a specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), an HCDR2, an HCDR3, a light chain complementarity determining region 1 (LCDR1), an LCDR 2 and an LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 93, 94, 95, 123, 124 and 125, respectively; (2) SEQ ID NOs: 81, 82, 83, 111, 112 and 113, respectively; (3) SEQ ID NOs: 84, 85, 86, 114, 115 and 116, respectively; (4) SEQ ID NOs: 87, 88, 89, 117, 118 and 119, respectively; (5) SEQ ID NOs: 90, 91, 92, 120, 121 and 122, respectively; (6) SEQ ID NOs: 96, 97, 98, 126, 127 and 128, respectively; (7) SEQ ID NOs: 99, 100, 101, 129, 130 and 131, respectively; (8) SEQ ID NOs: 102, 103, 104, 132, 133 and 134, respectively; (9) SEQ ID NOs: 105, 106, 107, 135, 136 and 137, respectively; or (10) SEQ ID NOs: 108, 109, 110, 138, 139 and 140, respectively; wherein the antibody or antigen binding fragment thereof specifically binds to CLDN18.2, preferably to human CLDN18.2.
[0070] [0070] According to another specific aspect, the invention relates
[0071] [0071] According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment of the same invention comprising: a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; B. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; ç. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4; d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 6; and. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8; f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12;
[0072] [0072] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 21, 22, 23, 51, 52 and 53, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a variable region of heavy chain with a polypeptide sequence of at least 85%, preferably 90%, more preferably
[0073] [0073] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 24, 25, 26, 54, 55 and 56, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 3; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4.
[0074] [0074] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LOCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 27, 28, 29, 57,
[0075] [0075] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 30, 31, 32, 60, 61 and 62, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8.
[0076] [0076] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 33, 34, 35, 63, 64 and 65, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10.
[0077] [0077] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LOCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 36, 37, 38, 66, 67 and 68, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12.
[0078] [0078] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 39, 40, 41, 69, 70 and 71, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14.
[0079] [0079] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 42, 43, 44, 72, 73 and 74, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID
[0080] [0080] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 45, 46, 47.75, 76 and 77, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18.
[0081] [0081] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 48, 49, 50, 78,
[0082] [0082] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LOCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 81, 82, 83, 111, 112 and 113, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2.
[0083] [0083] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 84, 85, 86, 114, 115 and 116, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 3; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4.
[0084] [0084] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 87, 88, 89, 117, 118 and 119, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 5; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 6.
[0085] [0085] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 90, 91, 92, 120, 121 and 122, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8.
[0086] [0086] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 93, 94, 95, 123, 124 and 125, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID
[0087] [0087] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 96, 97, 98, 126, 127 and 128, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12.
[0088] [0088] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LOCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 99, 100, 101, 129, 130 and 131, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment of the same comprises a variable region of heavy chain with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14.
[0089] [0089] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 102, 103, 104, 132, 133 and 134, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment of the same comprises a variable region of heavy chain with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more like 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16.
[0090] [0090] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 105, 106, 107, 135, 136 and 137, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment of the same comprises a variable region of heavy chain with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18.
[0091] [0091] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs : 108, 109, 110, 138, 139 and 140, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment of the same comprises a variable region of heavy chain with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, and a light chain variable region with a polypeptide sequence of at least 85%, preferably 90%, with more preferably 95% or more, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20. Preferably, the isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19; and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20.
[0092] [0092] According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen binding fragment of the same invention, wherein the antibody or antigen binding fragment thereof is chimeric.
[0093] [0093] According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment of the same invention, wherein the antibody or antigen-binding fragment thereof is human or humanized.
[0094] [0094] According to another specific aspect, the invention relates to an isolated humanized monoclonal antibody or antigen-binding fragment thereof, wherein the isolated humanized antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; B. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; ç. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; and. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148;
[0095] [0095] In another general aspect, the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof. It will be understood by those skilled in the art that a protein's coding sequence can be altered (for example, substituted, deleted, inserted, etc.) without altering the amino acid sequence of the protein. Thus, it will be understood by those skilled in the art that nucleic acid sequences encoding monoclonal antibodies or antigen-binding fragments thereof can be altered without changing the amino acid sequences of proteins.
[0096] [0096] In another general aspect, the invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof. Any vector known to those skilled in the art in view of the present disclosure can be used, as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector like a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. Various expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used here for the production of an antibody or antigen binding fragment thereof in the cell. Conventional cloning techniques or synthesis of artificial genes can be used to generate a recombinant expression vector according to the modalities of the invention.
[0097] In another general aspect, the invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof. Any host cell known to those skilled in the art in view of the present disclosure can be used for the recombinant expression of antibodies or antigen-binding fragments thereof of the invention. In some embodiments, the host cells are E. coli TG1 or BL21 cells (for expression of, for example, an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells or HEK293 cells (for expression of, for example , a full-length IgG antibody). According to specific modalities, the recombinant expression vector is transformed into host cells by conventional methods, such as chemical transfection, heat shock or electroporation, where it is stably integrated into the host cell's genome, so that the acid recombinant nucleic acid is effectively expressed.
[0098] [0098] In another general aspect, the invention relates to a method for producing a monoclonal antibody or antigen-binding fragment of the same invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or the antigen binding fragment thereof, capable of producing a monoclonal antibody or the antigen binding fragment thereof, and recovering the antibody or antigen binding fragment thereof from the cell or cell culture (for example, example, of the supernatant). The expressed antibodies or antigen-binding fragments thereof can be harvested from cells and purified according to conventional techniques known in the art and described herein. Pharmaceutical compositions
[0099] [0099] In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment of the invention and a pharmaceutically acceptable carrier. The term "pharmaceutical composition", as used herein, means a product comprising an antibody of the invention together with a pharmaceutically acceptable carrier. The antibodies of the invention and the compositions that comprise them are also useful in the manufacture of a medicament for therapeutic applications mentioned here.
[0100] [0100] As used here, the term "vehicle" refers to any excipient, diluent, charge, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the vehicle,
[0101] [0101] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, Remington: he Science and Practice of Pharmacy (for example, 21st edition (2005) and any later edition). Non-limiting examples of additional ingredients include: buffers, thinners, solvents, tonicity regulating agents, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used in the formulation of the pharmaceutical compositions of the invention.
[0102] [0102] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, that is, a formulation composed of water. The liquid formulation can comprise a solution, a suspension, an emulsion, a microemulsion, a gel and the like. An aqueous formulation generally comprises at least 50% water w / w, or at least 60%, 70%, 75%, 80%, 85%, 90% or at least 95% w / w water.
[0103] [0103] In one embodiment, the pharmaceutical composition can be formulated as an injectable that can be injected, for example, by means of an injection device (for example, a syringe or an infusion pump). The injection can be administered subcutaneously, intramuscularly, intraperitoneally, intravitreally or intravenously, for example.
[0104] [0104] In another embodiment, the pharmaceutical composition is a solid formulation, for example, a freeze-dried or spray-dried composition, which can be used as is, or to which the doctor or patient adds solvents and / or thinners before use. Solid dosage forms may include tablets, such as compressed tablets and / or coated tablets and capsules (for example, hard or soft gelatin capsules). The pharmaceutical composition can also be in the form of sachets, dredges, powders, granules, lozenges or powders for reconstitution, for example.
[0105] [0105] Dosage forms can be immediate release, in which case they can comprise a water soluble or dispersible vehicle, or they can be delayed release, sustained release or modified release, in which case they can include water insoluble polymers that regulate the rate of dissolution of the pharmaceutical form in the gastrointestinal tract or under the skin.
[0106] [0106] In other modalities, the pharmaceutical composition can be applied intranasally, intrabucalally or sublingually.
[0107] [0107] The pH in an aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is about 3.0 to about 7.0.
[0108] [0108] In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicin, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, malic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine and tris (hydroxymethyl) -aminomethane and mixtures thereof. The buffer may be present individually or in the aggregate, or at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the invention.
[0109] [0109] In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol , ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservative can be present individually or in the aggregate, or at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments of the invention.
[0110] [0110] In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of modality include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and trionine), an alditol (such as glycerol, 1,2-propanediol propylene glycol!), 1,3-propanediol, and 1,3-butanediol), polyethylene glycol (e.g., PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars can be mono-, di- or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethyl cellulose. Another example of an isotonic agent is a sugar alcohol, in which the term "sugar alcohol" is defined as a C (4-8) hydrocarbon with at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol. The isotonic agent can be present individually or in the aggregate, or a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml . Pharmaceutical compositions comprising each of these specific isotonic agents are alternative embodiments of the invention.
[0111] [0111] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylene diaminetetraacetic acid (EDTA) and mixtures thereof. The chelating agent can be present individually or in the aggregate, or a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the invention.
[0112] [0112] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants and / or one or more protease inhibitors.
[0113] [0113] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy / hydroxyculose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2 -methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulfur-containing substances, such as monothioglycerol, or thioglycolic acid. The stabilizer may be present individually or in the aggregate, or a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, from about 0.1 mg / ml to about 20 mg / ml.
[0114] [0114] In additional embodiments of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecules or ions that are composed of a water-soluble part (hydrophilic) and a fat-soluble part (lipophilic). The surfactant can, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, non-ionic surfactants and / or zwitterionic surfactants. The surfactant can be present individually or in the aggregate at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the invention.
[0115] [0115] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and / or benzamidine hydrochloric acid (HCI). The protease inhibitor can be present individually or in the aggregate at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions composed of each of these specific protease inhibitors are alternative embodiments of the invention.
[0116] [0116] In another general aspect, the invention relates to a method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment of the same invention, the combination comprising a monoclonal or fragment-binding antibody antigen with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. Usage methods
[0117] [0117] In another general aspect, the invention relates to a method of targeting CLDN18.2 on a cancer cell surface in an individual to achieve cell death, the method comprises administering to the individual a monoclonal antibody isolate or an antigen-binding fragment thereof that specifically binds to CLDN18.2 or a pharmaceutical composition comprising the isolated monoclonal antibody or the antigen-binding fragment of the invention. Binding of the CLDN18.2 monoclonal antibody or antigen-binding fragment to CLDN18.2 can mediate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC) and / or antibody-dependent cell cytotoxicity (ADCC) or other effects that result in the death of the target cancer cell. The monoclonal antibody or antigen-binding fragment thereof may, for example, serve to recruit conjugated drugs and / or may form a bispecific antibody with another monoclonal antibody to mediate the death of the target cancer cell.
[0118] [0118] The functional activity of antibodies and antigen-binding fragments thereof that bind CLDN18.2 can be characterized by methods known in the art and as described here. Methods for characterizing antibodies and antigen-binding fragments that bind to CLDN18.2 include, but are not limited to, affinity and specificity tests, including Biacore, ELISA and OctetRed analysis, and detection of binding from antibodies and antigen-binding fragments to CLDN18.2 in cells (either cells transfected with CLDN18.2 or cells that naturally express CLDN18.2) by FACS. According to specific modalities, the methods for characterizing antibodies and antigen-binding fragments thereof that bind CLDN18.2 include those described below.
[0119] [0119] In another general aspect, the invention relates to a method for treating cancer in an individual who needs it, comprising administering to the individual an isolated monoclonal antibody or an antigen-binding fragment of the same that binds specifically CLDN18.2 or a pharmaceutical composition of the invention. Cancer can, for example, be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, carcinoma of kidney cells, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblasma and other solid tumors, and non-Hodgkin's lymphoma (NHL ), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.
[0120] [0120] In another general aspect, the invention relates to a method for treating an inflammatory disease to an individual who needs it, comprising administering to the individual an isolated monoclonal antibody or an antigen-binding fragment of the same that specifically binds CLDN18.2 or a pharmaceutical composition of the invention.
[0121] [0121] According to the modalities of the invention, the pharmaceutical composition comprises an effective therapeutic amount of an anti-CLDN18.2 antibody or antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in an individual. The therapeutically effective amount can be determined empirically and routinely, in relation to the stated objective.
[0122] [0122] As used herein with reference to anti-CLDN18.2 antibodies or antigen binding fragments thereof, a therapeutically effective amount means an amount of the anti-CLDN18.2 antibody or antigen binding fragment thereof that modulates an immune response in an individual who needs it. In addition, As used herein with reference to anti-CLDN18.2 antibodies or antigen binding fragments thereof, a therapeutically effective amount means an amount of the anti-CLDN18.2 antibody or antigen binding fragment thereof. that results in the treatment of a disease, disorder or condition; prevents or delays the progression of the disease, disorder or condition; or completely reduces or relieves symptoms associated with the disease, disorder or condition.
[0123] [0123] According to specific modalities, the disease, disorder or condition to be treated is cancer, preferably a cancer selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, cancer of the bile duct, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial bladder carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma , glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML ) and other liquid tumors. According to other specific modalities, the disease, disorder or condition to be treated is an inflammatory disease.
[0124] [0124] According to specific modalities, a therapeutically effective amount refers to the amount of therapy that is sufficient to achieve one, two, three, four or more of the following effects: (i) reduce or improve the severity of disease, disorder or condition being treated or a symptom associated with it; (ii) reducing the duration of the disease, disorder or condition to be treated or a symptom associated with it; (iii) preventing the progression of the disease, disorder or condition to be treated, or a symptom associated with it; (iv) cause the disease, disorder or condition to be treated to regress, or a symptom associated with it; (v) prevent the development or appearance of the disease, disorder or condition to be treated, or a symptom associated with it; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated with it; (vii) reduce the hospitalization of an individual who has the disease, disorder or condition to be treated, or a symptom associated with it; (viii) reduce the length of hospital stay of an individual who has the disease, disorder or condition to be treated, or a symptom associated with it; (ix) increase the survival of an individual with the disease, disorder or condition to be treated, or a symptom associated with it; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated with it; and / or (xii) improve or accentuate the prophylactic or therapeutic effect (s) of another therapy.
[0125] [0125] The therapeutically effective amount or dosage may vary according to several factors, such as the disease, disorder or condition to be treated, the means of administration, the target location, the individual's physiological state (including, for example , age, body weight, health), whether the individual is human or animal, other medications administered and whether the treatment is prophylactic or therapeutic. Treatment dosages are ideally titrated to optimize safety and effectiveness.
[0126] [0126] According to specific modalities, the compositions described here are formulated to be suitable for the intended route of administration for an individual. For example, the compositions described here can be formulated to be suitable for intravenous, subcutaneous or intramuscular administration.
[0127] [0127] As used here, the terms "treat", "treating" and "treatment" are intended to refer to an improvement or reversal of at least one measurable physical parameter related to cancer and / or inflammatory disease , a disorder or condition, which is not necessarily perceptible in the individual, but can be perceptible in the individual. The terms "treat", "treating and" treatment "can also refer to causing regression, preventing progression or, at least, slowing the progression of the disease, disorder or condition. In a particular mode," treat "," treating "and" treatment "refer to relief, prevention of development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder or condition, such as a tumor or more preferably In a specific embodiment, "treating", "treating" and "treatment" refer to preventing the recurrence of the disease, disorder or condition. In a specific embodiment, "treating", "treating" and "treatment" "refer to an increase in the survival of an individual who has the disease, disorder or condition. In a specific modality," treat "," treating "and" treatment "refer to the elimination of the disease, disorder or condition in the individual.
[0128] [0128] In accordance with specific modalities, compositions used to treat cancer and / or an inflammatory disease, a disorder or a condition are provided. For cancer therapy, the compositions provided may be used in combination with another treatment, including, but not limited to, chemotherapy, an anti-CD20 mAb, an anti-TIM-3 mAb, an anti-LAG-3 mAb, an anti-EGFR mAb, an anti-HER-2 mAb, an anti-CD19 mAb, an anti-CD33 mAb, an anti-CD47 mAb, an anti-DLL-3 mAb, an anti-apelin mAb, an anti-TIP mAb -1, an anti-FOLR1 mAb, an anti-CTLA-4 mAb, an anti-PD-L1 mAb, an anti-PD-1 mAb, other immuno-cancer drugs, an anti-angiogenic agent, radiation therapy, an antibody- drug (ADC), a target therapy or other anticancer drugs. Anti-CLDN18.2 antibodies can be used to build bispecific antibodies with partner mAbs against PD-1, PD-L1, LAG3, TIM-3, CTLA-4,
[0129] [0129] As used here, the term "in combination", in the context of administering two or more therapies to an individual, refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to an individual. For example, a first therapy (for example, a composition described here) can be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before), concomitantly with, or subsequent to (eg 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week , 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administering a second therapy to an individual.
[0130] [0130] In another general aspect, the invention relates to a method of determining a level of CLDN18.2 in an individual. Methods include (a) obtaining a sample from the individual; (b) placing the sample in contact with a monoclonal antibody or antigen-binding fragment of the invention; and (c) determining a level of CLDN18.2 in the individual.
[0131] [0131] As used herein, "sample" refers to a biological sample isolated from an individual and may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies ( for example, cancer tissue), lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus,
[0132] [0132] In certain embodiments, the level of CLDN18.2 in the individual can be determined using assays selected from, but not limited to, a Western blot, immunohistochemistry (IHC) assay and an ELISA assay. The relative levels of protein can be determined using Western blot and IHC analysis, and the absolute levels of protein can be determined using an ELISA assay. When determining the relative levels of CLDN18.2, the levels of CLDN18.2 can be determined between at least two samples, for example, between samples from the same individual at different time points, between samples of different tissues in the same individual and / or between samples from different individuals. Alternatively, by determining the absolute levels of CLDN18.2, as with an ELISA assay, the absolute level of CLDN18.2 in the sample can be determined by creating a standard for the ELISA assay before testing the sample. One skilled in the art will understand which analytical techniques to use to determine the level of CLDN18.2 in a sample from the subject using the antibodies or antigen-binding fragments of the subject of the invention.
[0133] [0133] The use of methods to determine a level of CLDN18.2 in a sample of an individual can lead to the diagnosis of abnormal (high, reduced or insufficient) levels of CLDN18.2 in a disease and to make appropriate therapeutic decisions. This disease can be selected, but is not limited to, a cancer and an inflammatory disease. In addition, by monitoring the levels of CLDN18.2 in an individual, the risk of developing a disease as indicated above can be determined based on knowledge of the level of CLDN18.2 in a specific disease and / or during the progression of the disease. - specific task. MODALITIES
[0134] [0134] The invention also provides the following non-limiting modalities.
[0135] [0135] Mode 1 is an isolated monoclonal antibody or antigen binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR 2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 33, 34, 35, 63, 64 and 65, respectively; (2) SEQ ID NOs: 21, 22, 23, 51, 52 and 53, respectively; (3) SEQ ID NOs: 24, 25, 26, 54, 55 and 56, respectively; (4) SEQ ID NOs: 27, 28, 29, 57, 58 and 59, respectively; (5) SEQ ID NOs: 30, 31, 32, 60, 61 and 62, respectively; (6) SEQ ID NOs: 36, 37, 38, 66, 67 and 68, respectively; (7) SEQ ID NOs: 39, 40, 41, 69, 70 and 71, respectively; (8) SEQ ID NOs: 42, 43, 44, 72, 73 and 74, respectively; (9) SEQ ID NOs: 45, 46, 47, 75, 76 and 77, respectively; or (10) SEQ ID NOs: 48, 49, 50, 78, 79 and 80, respectively; wherein the antibody or antigen binding fragment thereof specifically binds to claudin 18.2 (CLDN18.2), preferably specifically binds to human CLDN18.2.
[0136] [0136] Mode 2 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 ( LCDR1), LCDR2 and LCDR3, with the polypeptide sequences of: (1) SEQ ID NOs: 93, 94, 95, 123, 124 and 125, respectively; (2) SEQ ID NOs: 81, 82, 83, 111, 112 and 113, respectively;
[0137] [0137] Mode 3 is the isolated monoclonal antibody or antigen binding fragment of mode 1 or 2, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 9 , 1, 3, 5, 7, 11, 13, 15, 17 or 19, or a light chain variable region with a polypeptide sequence of at least 95% identical to SEQ ID NO: 10,2,4,6, 8, 12, 14,16, 18 or 20.
[0138] [0138] Mode 4 is the isolated monoclonal antibody or antigen binding fragment of mode 1 or 2, comprising (a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a variable region light chain having the polypeptide sequence of SEQ ID NO: 10; (b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2;
[0139] [0139] Mode 5 is the isolated monoclonal antibody or antigen-binding fragment of any of modalities 1 to 4, wherein the monoclonal antibody or the antigen-binding fragment thereof is capable of inducing effector-mediated tumor cell lysis .
[0140] [0140] Mode 6 is the isolated monoclonal antibody or the antigen-binding fragment of any of modalities 1 to 5,
[0141] [0141] Mode 7 is the isolated monoclonal antibody or the antigen-binding fragment of any one of modalities 1 to 6, wherein the antibody or the antigen-binding fragment of the same is human or humanized.
[0142] [0142] Mode 8 is the isolated monoclonal antibody or antigen-binding fragment of mode 7, in which the isolated human antibody or antigen-binding fragment of the same comprises: a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; B. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; ç. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; and. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148; f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 149; g. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150;
[0143] [0143] Mode 9 is the isolated monoclonal antibody or antigen-binding fragment of any of modalities 1 to 8, in which the isolated humanized antibody or antigen-binding fragment thereof is capable of inducing effector-mediated tumor cell lysis through antibody-dependent cell cytotoxicity (ADCC), antibody-dependent phagocytosis (ADPC) and / or complement-dependent cytoxicity (CDC), and / or mediating the recruitment of conjugated drugs, and / or forming a bispecific antibody with another mAb or antigen-binding fragment of the same to kill cancer.
[0144] [0144] Mode 10 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof, according to any of modalities 1 to 9.
[0145] [0145] Mode 11 is a vector comprising isolated nucleic acid of mode 10.
[0146] [0146] Mode 12 is a host cell comprising the mode 11 vector.
[0147] [0147] Mode 13 is a pharmaceutical composition comprising
[0148] [0148] Modality 14 is a method for targeting CLDN18.2 on a cancer cell surface in an individual who needs it, comprising administering to the individual a pharmaceutical composition of modality 13.
[0149] [0149] Modality 15 is a method of treating cancer in an individual who needs it, including administering to the individual the pharmaceutical composition of modality 13.
[0150] [0150] Modality 16 is the method of modality 15, in which cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, carcinoma kidney cells, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), leukemia acute lymphocytic (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.
[0151] [0151] Mode 17 is a method to treat an inflammatory disease in an individual who needs it, comprising the administration to the individual of a pharmaceutical composition of the modality 13.
[0152] [0152] Mode 18 is a method for producing the monoclonal antibody or antigen-binding fragment of any of modalities 1 to 9, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or the antigen-binding fragment thereof, capable of producing the monoclonal antibody or the antigen-binding fragment thereof,
[0153] [0153] Mode 19 is a method for producing a pharmaceutical composition comprising the monoclonal antibody or an antigen binding fragment of modalities 1 to 9, comprising the combination of the monoclonal antibody or the binding fragment the antigen thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
[0154] [0154] Mode 20 is a method for determining a level of CLDN18.2 in an individual, the method comprising: a. obtain a sample from the individual; B. placing the sample in contact with the isolated monoclonal antibody or antigen-binding fragment of any of modalities 1 to 9; and c. determine a level of CLDN18.2 in the individual.
[0155] [0155] Mode 21 is the method of mode 20, in which the sample is a tissue sample.
[0156] [0156] Mode 22 is the method of mode 21, in which the tissue sample is a cancer tissue sample.
[0157] [0157] Mode 23 is the method of mode 20, where the sample is a blood sample.
[0158] [0158] The mice were immunized with cells expressing exogenous human CLDN18.2 and the plasma titration was determined by the selection of fluorescence activated cells (FACS). Splenocytes were harvested and fused with a myeloma cell line to produce hybridomas. Hybridomas were coated on 384-well plates and supernatants from individual wells were screened by FACS using HEK293 cells expressing CLDN18.2. Positive clones were counterstained against human CLDN18.1 expressed in HEK293 cells by FACS. The upper positive clones were isolated and sequenced.
[0159] [0159] The sequences of heavy and light chain variable regions for anti-CLDN18.2 monoclonal antibodies are provided in Tables 1 and 2, and the CDR regions for anti-CLDN18.2 monoclonal antibodies are provided in Tables 3-6 . Table 1: Sequences of heavy chain variable regions for anti-CLDN18.2 mAbs SEQ ID clones
[0160] [0160] HC: heavy chain; CDR: complementarity determining region; ID: SEQ ID NO
[0161] [0161] HC CDRs for anti-CLDN18.2 mAbs were determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).
[0162] [0162] LC: light chain; CDR: complementary determining region; NO: SEQ ID NO
[0163] [0163] LC CDRs for anti-CLDN18.2 mAbs were determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).
[0164] [0164] HC: heavy chain; CDR: complementarity determining region; NO: SEQ ID NO
[0165] [0165] HC CDRs for anti-CLDN18.2 mAbs have been determined using a combination of IMGT methods (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212) and Kabat (Elvin A. Kabat et al, Sequences of Proteins of Immunological Interest 5th ed. (1991)). Table 6: Light chain CDR 1-3 regions for anti-CLDN18.2 mAbs clones LC CDR1 NO | LCCDR2 LC CDR3 mAb number KSSQSLLNSGNOKNYLT [111 | WASTRES QNDYSYPLT [113 KASQDINRYLS 114 | RANRLVD | 115 | LQOYDEFPLT | 116 3-E21 KSSQOSLLNSGNOKNYLT | 117 | WASTRES | 118 | ONDYFYPLT 119 3-P21 KSSQOSLLNSGNOKNYLT | 120 | WASTRES | 121 | ONDYFYPLT | 122 5-E22 HARQNINVWLS 123 | KASNLHT [124 | QQGANYPLT | 125 6-111 KSSLSLLNSGNQKNYLT | 126 | WASTRES | 127 | ONAYSYPLT | 128 8-G12 KSSQSLLNSGNOKNYLT [129 | WASTRES QNAYIYPLT [131 10-10 | KSSQTLLNSGNQKNYLT | 132 | WASTRES QNDYFYPFT | 134 10-K2 - | KSSOSLLNSGNOKNYLT | 135 | WASTRES | / 136 | QNDYSYPFT | 137 15-D6 RSSQSLLNSGNQKSYLT WASTRES QNDYYYPFT
[0166] [0166] LC: light chain; CDR: complementary determining region; NO: SEQ ID NO
[0167] [0167] LC CDRs for anti-CLDN18.2 mAbs were determined using a combination of IMGT methods (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212) and Kabat (Elvin A. Kabat et al, Sequences of Proteins of Immunological Interest 5th ed. (1991)). Example 2: Production and purification of mAbs from transfected 293E cell culture media
[0168] [0168] To obtain recombinant chimeric anti-CLDN18.2 mAbs, the expression vectors containing the variable regions of the mouse (VH and VL) fused to the constant regions of the human IgG1 heavy chain and the light chain hood, respectively , were transiently transfected into 293E cells. The recombinant antibodies produced in the 293E cell suspension were purified by protein A affinity chromatography. Example 3: FACS binding analysis of purified anti-CLDN18.2 antibodies
[0169] [0169] HEK293 cells stably transfected with full-length human CLDN18.2 were transferred to a 96-well plate. Wax of 50,000 cells were incubated with purified chimeric anti-CLDN18.2 mAbs (variable regions of mouse mAbs fused to the constant regions of the human IgG1 heavy chain and the kappa light chain, respectively) at various concentrations for 15 minutes in ice. In some cases,
[0170] [0170] The binding of the chimeric anti-CLDN18.2 mAbs to HEK293 cells stably transfected with full-length human CLDN18.2 (pool of stable cells) was analyzed by FACS. The results for dose-dependent binding are provided in Figs. 1A-1D. The binding of the chimeric anti-CLDN18.2 mAbs to HEK293 cells stably transfected with full-length human CLDN18.2 and CLDN18.1 (stable cell pools), respectively, was assessed using 10 Nm mAbs using FACS. The results of the FACS connection analysis are provided in Figs. 2A-2E. The data indicates that these mAbs specifically bind to CLDN18.2 and do not bind to CLDN18.1.
[0171] [0171] Chimeric anti-CLDN18.2 mAbs were also analyzed with a stable HEK293 cell line expressing full-length human CLDN18.2 (HEK293-CLDN18.2) using FACS as described above, except that the Final detection was performed using F (ab ') 2 from goat anti-human IgG Fc conjugated to Alexa FluorO 488 (Invitrogen cat no: H10120). The results of the FACS analysis of the dose-dependent binding of the chimeric anti-CLDN18.2 mAbs to the HEK293-CLDN18.2 stable cell line are provided in Figs. 3A-3C. The histogram for the attachment of each 7.6 nM chimeric mAb to the stable HEK293-CLDN18.2 cell line is shown in Figs. 4A-4J. These data indicate that the chimeric mAbs specifically bind to the stable cell line of HEK293-CLDN18.2. Example 4: Humanization of anti-CLDN18.2 mAbs
[0172] [0172] The 2-C3, 5-E22, 6- J11,3-E21 and 3-P21 mice anti-CLDN18.2 mAbs have been humanized to reduce the potential for immunogenicity when used in human patients. The strings
[0173] [0173] The humanized VH and VL regions were fused to the constant regions of the human IgG1 heavy chain and the kappa light chain, respectively. Constructions corresponding to the mAb sequences were used for transient transfection in 293E or CHO cells and purified MAbs were analyzed for their ability to bind to HEK293 cells stably transfected with full-length human CLDN18.2 using FACS. EC50 values of humanized mAbs for binding of CLDN18.2 using a stable pool of HEK293-CLDN18.2 are summarized in Table 8.
[0174] [0174] The results for the dose-dependent binding of humanized mAbs to the HEK293-CLDN18.2 stable pool are shown in Figs. 5A-5G. FITC-based detection was used in the FACS experiments in Table 8 and Figs SA-5C; detection based on Alexa FluorO 488 was used in the FACS experiments in Figs. 5D-5G.
[0175] [0175] Results for dose-dependent binding of humanized anti-CLDN18.2 mAbs to a stable HEK293-CLDN18.2 cell line by FACS analysis are shown in Figs. 6A-6E. The binding of humanized anti-CLDN18.2 mAbs to the NUGC-4 cancer cell line at the mAb concentration of 685 nM was also analyzed
[0176] [0176] Antibody-dependent cellular cytotoxicity (ADCC) of mAbs was measured using the ADCC reporter bioassay core kit (Promega, cat. No. G7010) according to the protocol provided by the manufacturer. Soon, about 12,500 HEK293-CLDN18.2 cells per well were mixed with various concentrations of test antibodies in the ADCC assay buffer in a 96-well, half-area microplate (Corning-Costar, cat. 43696). Then, about 37,500 per well of ADCC bioassay effector cells were added to a final volume of 37.5 µl. and mixed. The plate was incubated at 37 ºC for 6 hours without shaking. To measure luciferase activity, 12.5 µl of the test mixture was removed from each well and 25 µl of the Bio-Glo luciferase test reagent was added. The plates were incubated at room temperature for 10 minutes with shaking. 30 ul per well of the mixture was transferred to a white plate (BRAND, cat. No. 781621) to measure luminescence on an EnvVision 2102 multimode plate reader. The results of ADCC activity from the five humanized mAbs and a chimeric mAb are shown in Fig 8A-8B. RLU, relative light unit. Table 7: Sequences of variable regions of light chain and heavy chain for humanized anti-CLDN18.2 mAbs pm creed 1º | 2-C3-H1 | GKALEWVATISGGGSYTYYNPSLKDRFTISRDISANQLVLKVT | 142
[0177] [0177] 2-C3-H1L1 refers to the mAb with the 2-C3-H1 heavy chain variable region and the 2-C3-L1 light chain variable region; all other humanized mAbs in the table and the text adopt the same nomenclature rule.
[0178] [0178] It will be understood by those skilled in the art that changes could be made in the modalities described above without departing from the broad inventive concept of them. It is understood, therefore, that this invention is not limited to the specific modalities disclosed, but aims to cover modifications within the spirit and scope of the present invention, as defined by the present description.

权利要求:
Claims (1)
[1]
1. Isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR 2 and LCDR3, with the polypeptide sequences of: a. SEQ ID NOs: 33, 34, 35, 63, 64 and 65, respectively; B. SEQ ID NOs: 21, 22, 23, 51, 52 and 53, respectively; ç. SEQ ID NOs: 24, 25, 26, 54, 55 and 56, respectively; d. SEQ ID NOs: 27, 28, 29, 57, 58 and 59, respectively; and. SEQ ID NOs: 30, 31, 32, 60, 61 and 62, respectively; f. SEQ ID NOs: 36, 37, 38, 66, 67 and 68, respectively; g. SEQ ID NOs: 39, 40, 41, 69, 70 and 71, respectively; H. SEQ ID NOs: 42, 43, 44, 72, 73 and 74, respectively; i. SEQ ID NOs: 45, 46, 47, 75, 76 and 77, respectively; or j. SEQ ID NOs: 48, 49, 50, 78, 79 and 80, respectively; characterized by the fact that the antibody or antigen-binding fragment thereof specifically binds to claudin 18.2 (CLDN18.2), preferably to human CLDN18.2.
2. Isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR 2 and LCDR3, with the polypeptide sequences of: a. SEQ ID NOs: 93, 94, 95, 123, 124 and 125, respectively; B. SEQ ID NOs: 81, 82, 83, 111, 112 and 113, respectively; ç. SEQ ID NOs: 84, 85, 86, 114, 115 and 116, respectively; d. SEQ ID NOs: 87, 88, 89, 117, 118 and 119, respectively; and. SEQ ID NOs: 90, 91, 92, 120, 121 and 122, respectively; f. SEQ ID NOs: 96, 97, 98, 126, 127 and 128, respectively;
g. SEQ ID NOs: 99, 100, 101, 129, 130 and 131, respectively; H. SEQ ID NOs: 102, 103, 104, 132, 133 and 134, respectively; i. SEQ ID NOs: 105, 106, 107, 135, 136 and 137, respectively; or j. SEQ ID NOs: 108, 109, 110, 138, 139 and 140, respectively; characterized by the fact that the antibody or antigen-binding fragment thereof specifically binds to claudin 18.2 (CLDN18.2), preferably to human CLDN18.2.
3. Isolated monoclonal antibody or antigen-binding fragment thereof, according to claim 1 or 2, characterized by the fact that it comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to that SEQ ID NO: 9, 1, 3, 5, 7, 11, 13, 15, 17 or 19, or a light chain variable region with a polypeptide sequence of at least 95% identical to SEQ ID NO: 10, 2 , 4, 6, 8, 12, 14, 16, 18 or 20.
4. Isolated monoclonal antibody or antigen-binding fragment thereof, according to claim 1 or 2, characterized by the fact that it comprises a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; B. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; ç. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4;
d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 6; and. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8; f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12;
9. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14; H. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; i. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18; j. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20;
5. Isolated monoclonal antibody or antigen-binding fragment thereof, according to any one of claims 1 to 4, characterized by the fact that the monoclonal antibody or the antigen-binding fragment thereof is capable of inducing lysis tumor cell mediated by the effector.
6. Isolated monoclonal antibody or antigen-binding fragment thereof, according to any one of claims 1 to 5, characterized in that the antibody or antigen-binding fragment is chimeric.
7. Isolated monoclonal antibody or antigen-binding fragment thereof, according to any one of claims 1 to 6, characterized in that the antibody or antigen-binding fragment thereof is human or humanized.
8. Isolated monoclonal antibody or antigen-binding fragment thereof, according to claim 7, characterized by the fact that the isolated humanized antibody or antigen-binding fragment thereof comprises: (a) a variable region of heavy chain having the polypeptide sequence of SEQ ID NO: 142 and a variable region of light chain having the polypeptide sequence of SEQ ID NO: 144; (b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; (c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144; (d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 143 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 145; (e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148; (f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 149; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150; (h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148;
(i) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 149;
(]) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 147 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 150;
(k) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 151 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 153;
(1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 152 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 153;
(m) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 154 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 157;
(n) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 157;
(0) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 156 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 158;
(p) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 163;
(q) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 159 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 164;
(r) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 163; (s) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 160 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 164; (t) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 161 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 165; or (u) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 165.
9. Isolated monoclonal antibody or antigen-binding fragment thereof, according to any one of claims 1 to 8, characterized in that the antibody or antigen-binding fragment thereof is capable of inducing tumor cell lysis mediated by effectors through antibody-dependent cell cytotoxicity (ADCCO), antibody-dependent phagocytosis (ADPC) and / or complement-dependent cytoxicity (CDC), and / or mediating the recruitment of conjugated drugs, and / or forming a bispecific antibody with another mAb or antigen-binding fragment of the same to kill cancer.
10. Isolated nucleic acid characterized by the fact that it encodes the monoclonal antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 9.
11. Vector characterized by the fact that it comprises isolated nucleic acid as defined in claim 10.
12. Host cell characterized by the fact that it comprises the vector, as defined in claim 11.
Pharmaceutical composition characterized in that it comprises the isolated monoclonal antibody or antigen-binding fragment thereof, as defined in any one of claims 1a9, and a pharmaceutically acceptable carrier.
14. Method for targeting CLDN18.2 on a cancer cell surface at an individual who needs it, characterized by the fact that it understands the administration to the individual of the pharmaceutical composition, as defined in claim 13.
15. Method for treating cancer in an individual who needs it, characterized by the fact that it understands the administration of the pharmaceutical composition to the individual, as defined in the claim
18.
16. Method, according to claim 15, characterized by the fact that the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, carci - renal cell noma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL ), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.
17. Method for treating an inflammatory disease in an individual who needs it, characterized by the fact that it comprises the administration to the individual of the pharmaceutical composition, as defined in claim 13.
18. Method for producing the monoclonal antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 9, characterized in that it comprises the culture of a cell comprising a nucleic acid encoding the monoclonal antibody or the antigen-binding fragment thereof, capable of producing the monoclonal antibody or the antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment from the cell or culture.
19. Method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment as defined in any one of claims 1 to 9, characterized in that it comprises the combination of the monoclonal antibody or the binding fragment the antigen thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
20. Method for determining a level of CLDN18.2 in an individual, characterized by the fact of understanding: a. obtain a sample from the individual; B. contacting the sample with an isolated monoclonal antibody or antigen-binding fragment thereof, as defined in any of claims 1a9; and c. determine a level of CLDN18.2 in the individual.
21. Method according to claim 20, characterized by the fact that the sample is a tissue sample.
22. Method according to claim 21, characterized by the fact that the tissue sample is a cancer tissue sample.
23. Method according to claim 20, characterized by the fact that the sample is a blood sample.

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法律状态:
2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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PCT/US2019/020872|WO2019173420A1|2018-03-08|2019-03-06|Anti-claudin 18.2 antibodies and uses thereof|

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