Tumor-specific claudin 18.2 antibody-drug conjugates

Abstract

The present invention provides an antibody-drug conjugate (ADC) using an antibody that binds to CLDN18.2, which antibody or a fragment thereof binds more strongly to tumor tissue expressing CLDN18.2 than to normal tissue expressing CLDN18.2.

Description

[Background technology]

[0001] Tight junctions are multiprotein complexes that connect adjacent epithelial and endothelial cells to form a barrier that prevents the passage of molecules between cells and helps maintain cell and tissue polarity. Tight junctions consist of three main groups of transmembrane proteins, claudins, occludin, and cingulin, of which claudins and occludin are intracellular plaque proteins. Tight junctions also contain cytoskeletal and signaling proteins, e.g., actin, myosin II, and PKCζ. These proteins interact to maintain the structure of the tight junctions (Yu and Turner, 2008).

[0002] Claudins constitute a family of 23 proteins (Hewitt, Agarwal, and Morin, 2006). Claudin 18 is a human protein encoded by the CLDN18 gene, which forms tight junction strands in epithelial cells. Human CLDN18 is produced by alternative splicing of the first two alternative exons, resulting in two protein isoforms, CLDN18.1 (claudin 18.1) and CLDN18.2 (claudin 18.2). CLDN18.2 was originally disclosed as the Zsig28 protein in WO2000 / 015659. These two isoforms differ in the amino acid sequence of the N-terminal 69 residues, which includes the first extracellular loop. This first extracellular domain consists of amino acids 28-80. In this amino acid sequence, CLDN18.1 and CLDN18.2 differ by eight amino acids. These two isoforms are expressed in different tissues. CLDN18.1 is mainly expressed in lung tissues, while CLDN18.2 is specifically expressed in the stomach (Niimi et al., 2001). CLDN18.2 expression in normal stomach is found only in differentiated short-lived gastric epithelial cells. In addition, CLDN18.2 expression has also been identified in various tumor tissues. For example, CLDN18.2 has been found to be expressed in pancreatic, esophageal, ovarian and lung tumors, and is associated with the presence of various histological subtypes (Sahin et al., 2008). The amino acid sequence of human CLDN18.2 protein has the NCBI reference sequence NP_001002026.1. This sequence may be derived from SEQ ID NO: 135.

[0003] CLDN18.2 has attracted attention as a cancer target for antibody therapy of epithelial tumors because its expression pattern in normal tissues is restricted to certain cells and it is ectopically expressed in human cancers. Various studies have been conducted to realize such antibody therapy. In WO2004 / 047863, splice variants of CLDN18 have been identified, and antibodies against various peptides derived from CLDN18.2 have been screened. In this screening, the following peptides derived from CLDN18.2 were used: a peptide consisting of DQWSTQDLYN (SEQ ID NO: 57) (the N-terminal extracellular domain of CLDN18.2, the antibody binds regardless of the presence or absence of glycosylation); a peptide consisting of NNPVTAVFNYQ (SEQ ID NO: 58) (the N-terminal extracellular domain of CLDN18.2, the antibody binds mainly to the non-glycosylated form); and a peptide consisting of STQDLYNNPVTAVF (SEQ ID NO: 59) (the N-terminal extracellular domain of CLDN18.2, the antibody binds only to the non-glycosylated form). Furthermore, this patent document also discloses polyclonal rabbit antibodies screened using a pan-CLDN18 peptide consisting of TNFWMSTANMYTG (SEQ ID NO: 60) contained in the C-terminal extracellular domain common to both the CLDN18.1 and CLDN18.2 isoforms. WO2005 / 113587 discloses antibodies against a specific epitope of CLDN18.2 defined by the peptide sequence ALMIVGIVLGAIGLLV (SEQ ID NO: 61) or RIGSMEDSAKANMTLTSGIMFIVS (SEQ ID NO: 62). WO2007 / 059997 discloses a CLDN18.2-specific monoclonal antibody obtained by immunization with a peptide consisting of METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLGTELGSTPVWWNSADGRMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRAAIQHSGGRSRRARTKTHLRRGSE (SEQ ID NO: 63), which contains the first extracellular domain of CLDN18.2 extended at the N-terminus and C-terminus.The antibodies obtained by this immunization exert cytotoxicity via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). WO2007 / 059997 and WO2016 / 165762 disclose an antibody called IMAB362, also known as Claudiximab or Zolbetuximab. IMAB362 is an IgG1 antibody derived from a mouse monoclonal antibody, and for clinical application, a human IgG1 constant region has been introduced by chimerization. WO2008 / 145338 discloses various antibodies that bind to overlapping peptides in the first extracellular domain (MDQWSTQDLYNNPVT (SEQ ID NO: 64), LYNNPVTAVFNYQGL (SEQ ID NO: 65), VFNYQGLWRSCVRES (SEQ ID NO: 66), QGLWRSCVRESSGFT (SEQ ID NO: 67) and RSCVRESSGFTECRG (SEQ ID NO: 68)). WO2013 / 167259 also attempts to produce an antibody targeting the C-terminal portion of CLDN18.2 in order to detect the expression of CLDN18.2 in cells in cancer tissue sections for diagnostic purposes, and discloses an antibody that binds to the C-terminal epitope of CLDN18.2. This patent document discloses two C-terminal epitopes of CLDN18.2, a sequence consisting of TEDEVQSYPSKHDYV (SEQ ID NO: 69) and a sequence consisting of EVQSYPSKHDYV (SEQ ID NO: 70). WO2013 / 174509 discloses a combination of an agent that stabilizes γδ T cells and an anti-CLDN18.2 antibody, and a combination of an agent that stabilizes or enhances the expression of CLDN18.2 and an anti-CLDN18.2 antibody. These anti-CLDN18.2 antibodies may be conjugated to a moiety having a therapeutic effect, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. WO2014 / 075788 discloses a method for treating cancer diseases using a bispecific antibody that binds to both CLDN18.2 and CD3. WO2014 / 127906 discloses the combination of multiple agents to stabilize or enhance the expression of CLDN18.2.WO2016 / 166122 discloses anti-CLDN18.2 monoclonal antibodies that can be internalized very efficiently when bound to CLDN18.2, and these characteristics make them suitable for the development of antibody-drug conjugates (ADCs). In addition, this patent also discloses the use of cleavable SPDB linkers or valine-citrulline linkers to link such antibodies to the drugs DM4 or MMAE. However, despite the various antibodies disclosed by these patent applications, the only one currently undergoing clinical trials is the chimeric antibody IMAB362 disclosed in WO2007 / 059997 and WO2016 / 165762. In addition to the aforementioned antibodies and antibody-drug conjugates, WO2018 / 006882 discloses chimeric antigen receptors (CARs) made from anti-CLDN18.2 monoclonal antibodies. In WO2018 / 006882, various antibodies are humanized, and the sequences of these humanized antibodies are disclosed in the supplementary materials of the collaborative research paper by Jiang et al. (2018) (Jiang et al., 2018). CAR T cells utilizing humanized antibodies are currently undergoing phase I clinical trials in patients with advanced gastric and pancreatic adenocarcinoma (ClinicalTrials.gov ID number: NCT03159819). In CN109762067, another anti-CLDN18.2 monoclonal antibody is disclosed that exerts cytotoxicity via CDC or ADCC. In WO2019 / 173420, an anti-CLDN18.2 humanized monoclonal antibody with ADCC activity is disclosed. In WO2019 / 175617, an anti-CLDN18.2 monoclonal antibody that binds to a different epitope than IMAB362 is disclosed. WO2019 / 219089 discloses a monoclonal antibody that binds to a mutant of CLDN18.2.Other antibodies that bind to CLDN18.2 are disclosed in WO2019 / 242505, WO2020 / 038404, WO2020 / 043044, WO2020 / 063988, WO2020 / 082209, WO2020 / 018852, WO2020 / 023679, WO2020 / 135674, WO2020 / 135201, WO2020 / 139956, WO2020 / 025792, WO2020160560, CN111808194, and WO2020200196.

[0004] CLDN18.2 has been reported to have various conformations and may contain N-glycosylation sites in the extracellular domain (see WO2007 / 059997, p. 3, paragraph 1). This suggests that the topology of CLDN18.2 may differ between normal and tumor cells, or that there may be differences in glycosylation sites (see WO2007 / 059997, p. 4, paragraph 2). However, no antibody has been reported to selectively target CLDN18.2 expressed in tumor cells. Since CLDN18.2 is expressed not only in tumors but also in normal tissues, i.e., gastric tissue (Sahin et al., 2008), it is clear that an antibody that targets only tumor-expressed CLDN18.2 would be beneficial to avoid the safety issues and side effects that often accompany the on-target effects of therapeutic antibodies on normal organs and tissues (Hansel et al., 2010), especially as reported for IMAB362 (Sahin et al., 2018; Tureci et al., 2019).

[0005] In addition to binding to the target with high affinity, therapeutic antibodies should retain the desired properties during development, manufacturing, storage and (in vivo) clinical application. Antibody stability can be compromised by post-translational modifications (PTMs) (Lu et al., 2019; Gervais, 2016). During the development of therapeutic antibodies, it is crucial to design therapeutic antibodies to undergo as few post-translational modifications as possible, since unregulated post-translational modifications can result in antibodies that do not meet the desired efficacy, activity, potency or stability. Post-translational modifications also have a significant impact on the regulatory acceptance, technology transfer or processing and development of biosimilars. The main modifications utilized are oxidation, deamidation and isomerization. Furthermore, IMAB362 is a chimeric antibody with an extended mouse sequence, which may induce anti-drug antibodies in patients, which may reduce the therapeutic efficacy, for example, when administered repeatedly.

[0006] As mentioned above, IMAB362 has also been developed as an antibody-drug conjugate (ADC) conjugated with the drug MMAE or DM4 (disclosed in WO2016 / 165762). For example, a conjugation of the drug DM4 and IMAB362 via SPBD (N-succinimidyl-3-(2-pyridyldithio)butyrate), a heterobifunctional protein crosslinker with amino and sulfhydryl reactivity, has been reported. SPBD reacts with primary amines of antibodies (present in lysine side chains and the N-terminus of proteins) via N-hydroxysuccinimide (NHS) ester. A conjugation of thiolated IMAB362 with valine-citrulline-MMAE (drug) has also been reported. In this case, IMAB362 is first thiolated with the heterobifunctional linker 2-IT (2-iminothiolane), which reacts with the free amine of lysine residues. Valine-citrulline is a cathepsin-cleavable linker. Note that all of the precautions regarding the IMAB362 mentioned above also apply to ADCs using the IMAB362.

[0007] Thus, there is a need for improved antibodies and ADCs specific for CLDN18.2 for use in treating tumor patients. Summary of the Invention

[0008] Definition of Terms An "antibody", also called an "immunoglobulin (Ig)", is a multimeric protein that typically contains four polypeptide chains, two heavy (H) and two light (L) chains. An "antibody" may also include immunoglobulin homologs, which are equivalent to immunoglobulins (e.g., camelid antibodies, which contain only heavy chains, and single domain antibodies (sdAbs) or nanobodies derived from heavy or light chains). An "antibody" also includes antibody-based binding proteins and recombinant antibody formats that retain the ability to bind to a target. An "antibody" also includes functional full-length mutants or variants or derivatives thereof that retain the epitope binding properties of the immunoglobulin molecule required for antibodies, including, but not limited to, murine, chimeric, humanized and fully human antibodies, including bispecific, bispecific, multispecific and bispecific variable domain immunoglobulins. The immunoglobulin molecules may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and any allotype. Immunoglobulin molecules may be mutated to improve or decrease affinity for, for example, Fcγ receptors or fetal Fc receptors (FcRn).

[0009] As used herein, an "antibody fragment" refers to a molecule that contains at least one polypeptide chain derived from an antibody, which molecule exhibits target binding properties but is not a full-length antibody. An antibody fragment can bind to the same epitope or the same target as the corresponding full-length antibody. Specific examples of antibody fragments include: (i) a Fab fragment (a monovalent fragment consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant domain (CL) and a heavy chain constant domain 1 (CH1)); (ii) a F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region (reducing the F(ab')2 fragment gives two Fab' fragments and a free sulfhydryl group); (iii) a heavy chain portion of a Fab (Fa) fragment (consisting of a VH domain and a CH1 domain); (iv) a variable fragment (Fv) (consisting of a VL domain and a VH domain in one arm of an antibody); (v) a domain antibody (dAb) fragment (containing one variable domain); (vi) an isolated complementarity determining region (CDR); (vii) a single chain Fv fragment (scFv); (viii) These include, but are not limited to, diabodies (bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, and the VH and VL domains are paired on the same polypeptide chain using a very short linker to pair with complementary domains in another chain to form two antigen-binding sites); (ix) linear antibodies (a pair of antigen-binding regions formed by pairing a linearly linked Fv segment (VH-CH1-VH-CH1) with a complementary light chain polypeptide); (x) immunoglobulins containing dual variable domains; and (xi) other fragments or portions of immunoglobulin heavy and / or light chains, or mutants, variants, or derivatives thereof, used alone or in combination.

[0010] As used herein, an "antibody-based binding protein" may refer to any protein that contains at least one VH, VL, or CH immunoglobulin domain derived from an antibody in a non-immunoglobulin or non-antibody derived component. Such antibody-based proteins include, but are not limited to, (i) fusion proteins of a binding protein with an Fc portion (e.g., a receptor or receptor component that contains all or a portion of the CH domains constituting an immunoglobulin), (ii) binding proteins in which the VH and / or VL domains are attached to another molecular scaffold, or (iii) molecules that combine and / or are constructed from immunoglobulin VH, VL, and / or CH domains in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0011] As used herein, "recombinant antibody formats" encompass antibody drug conjugates (ADCs), polyalkylene oxide modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bispecific or trispecific antibodies, IgA structures or structures consisting of two IgGs linked by a J chain and secretory component, shark antibodies, antibodies consisting of framework regions from a New World primate and CDRs from Old World primates, IgG4 antibodies with the hinge region removed, IgGs incorporating two additional binding sites in the CH3 domain, antibodies with modified Fc regions to improve or decrease affinity for Fcγ receptors, dimerization constructs comprising CH3, VL and VH, etc.

[0012] The Kabat numbering scheme (Martin and Allemn, 2014) has been applied to the antibodies disclosed herein.

[0013] An "antibody drug conjugate" or "ADC" refers to an antibody or antibody fragment to which a toxic drug (or drugs) is linked. The toxic drug in an ADC is linked to the antibody or antibody fragment by a cleavable or non-cleavable linker. Cleavable linkers can be designed to be cleaved extracellularly in the tumor environment or intracellularly in the cytoplasm. Cleavable linkers exploit the differential reducing power or enzymatic degradation conditions that may exist outside or inside the target cell. Non-cleavable linkers require the ADC to be internalized inside the cell, and the antibody-linker component must be degraded by lysosomal proteases to release the toxic drug. The linker-antibody bond can also vary. The bond between the antibody and the linker is determined by the presence of lysine and cysteine ​​residues in the polypeptide structure of the antibody, which serve as the attachment points between the two. The reactive groups on the linker can be attached, for example, by forming amide or amidine bonds with the side chains of the lysine residues. Attachment via cysteine ​​residues requires partial reduction of the antibody. Site-specific attachment by enzymes can also be used. In this case, an enzyme that reacts with the antibody and performs site-specific or amino acid sequence-specific modification is required, and the peptide sequence recognized by such an enzyme must be inserted into the recombinantly produced antibody or fragment. Enzymes used for this purpose include sortases, transglutaminases, galactosyltransferases, sialyltransferases, and tubulin-tyrosine ligases. A review of ADC linker conjugation and toxic drugs is provided in Ponziani et al. (Ponziani et al., 2020). A review of antibody fragment and toxic drug conjugation is provided in Aguiar et al. (Aguiar et al., 2018). It is believed that the drug-antibody ratio (DAR) is determined by the type of linker and conjugation method used to conjugate the toxic drug with the antibody or antibody fragment.

[0014] "Toxic drugs" refer to cytotoxic and / or cytostatic agents using synthetic, plant, fungal or bacterial molecules. Cytotoxic or cytostatic refers to inhibiting the proliferation and / or replication of cells, particularly malignant cells, usually malignant cells with increased turnover, and / or killing the cells. In a preferred embodiment, the toxic drug is selected from the group consisting of anthracyclines and their derivatives. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity and are believed to kill cells by various mechanisms, including the inhibition of DNA-dependent nucleic acid synthesis by the intercalation of the drug molecule into intracellular DNA or DNA cleavage activity, the generation of free radicals that react with cellular macromolecules to damage the cells, the alkylation of DNA, and / or the interaction of the drug molecule with the cell membrane. Anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and their derivatives. Among them, a well-known and preferred anthracycline derivative is PNU-159682, abbreviated as PNU, with CAS number 202350-68-3. PNU is a highly active metabolite of nemorubicin with excellent cytotoxicity. Anthracycline derivatives are understood to include anthracyclines bound to specific ligands, but the binding reaction may result in the loss of some atoms of the original anthracycline (Broggini, 2008 and Quintieri et al., 2005). In some cases, anthracycline derivatives can also be understood as lysosomal decomposition products in which a fragment of a linker remains bound to the anthracycline molecule. In this specification, "anthracycline" refers to anthracyclines and anthracycline derivatives.

[0015] As used herein, "selectively binds to CLDN18.2" or "selectively binds to CLDN18.2" refers to an antibody that has binding affinity to CLDN18.2 but does not have (specific) binding affinity to CLDN18.1. Thus, an antibody that selectively binds to CLDN18.2 does not exhibit cross-reactivity to CLDN18.1.

[0016] In the detailed description and claims of the present invention, when the term "comprising" is used, it does not exclude other elements. For the purposes of the present invention, an aspect described using the term "consisting of" is considered to be a preferred embodiment of the aspect described using the term "comprising". In the following, when a particular group is defined to include at least a certain number of embodiments, it is understood that a group preferably consisting of only these embodiments is also disclosed.

[0017] Where an indefinite or definite article is used for a singular noun, for example "a", "an" or "the", the plural of that singular noun is also included unless specifically stated otherwise.

[0018] Technical terms are used in their ordinary sense. If a particular term denotes a particular meaning, the definition of that term is set forth below in the context in which the term is used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present inventors have surprisingly identified, as detailed herein, novel antibody-drug conjugates (ADCs) comprising an anti-CLDN18.2 antibody and a toxic drug, which bind more strongly to CLDN18.2-expressing tumor cells than to CLDN18.2-expressing normal gastric cells, and / or have increased stability, and / or are humanized while retaining improved properties.

[0020] The present invention provides an ADC using an antibody that binds to CLDN18.2, characterized in that the antibody or a fragment thereof binds more strongly to tumor tissue expressing CLDN18.2 than to normal tissue expressing CLDN18.2. In one embodiment, the normal cell or tissue to be compared is a normal gastric cell or normal gastric tissue.

[0021] The strong binding of the antibodies or fragments thereof provided herein to tumor tissue may be confirmed by biological assays such as flow cytometry (FC) as shown in Example 4 and immunohistochemistry (IHC) as shown in Example 5. Tumors expressing CLDN18.2 may be generated by subcutaneously injecting A549 cells expressing CLDN18.2 into Balb / c mice. A549 cells expressing CLDN18.2 may be generated as described in Example 4 and are also available under accession number DSM ACC3360, deposited on December 6, 2019 at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B 38124 Braunschweig, Germany). Normal tissue (e.g., normal stomach tissue) may be collected from mice in which tumors were formed by the aforementioned subcutaneous injection. Thus, stronger binding to tumor tissue than normal tissue may be confirmed using tumor tissue and normal tissue obtained from the same animal.

[0022] The stronger binding to CLDN18.2 expressed in tumor tissues than to CLDN18.2 expressed in normal tissues may be due to post-translational modifications such as selective glycosylation of CLDN18.2 or misfolding of CLDN18.2.

[0023] The binding of the antibody may be evaluated using flow cytometry (FC) as a biological analysis method. The percentage of CLDN18.2 positive cells may be measured, for example, by flow cytometry using a specific anti-CLDN18.2 antibody. As another method for examining the binding of the antibody, for example, the ratio of the percentage of CLDN18.2 positive cells in a tumor cell sample to the percentage of CLDN18.2 positive cells in a cell sample obtained from a normal tissue (e.g., normal stomach tissue) may be evaluated. Stronger binding of the antibody to CLDN18.2-expressing tumor cells grown from A549 cells expressing CLDN18.2 than to normal cells (e.g., normal stomach cells) may be indicated by a ratio of more than 2, a ratio of more than 5, a ratio of 10 or more, preferably a ratio of 15 or more, and more preferably a ratio of 20 or more.

[0024] Stronger binding of an antibody to CLDN18.2-expressing tumor cells grown from A549 cells expressing CLDN18.2 than to normal cells (e.g., normal gastric cells) may be assessed by confirming that the number of tumor cells bound to the antibody is at least 2-fold, at least 5-fold, or at least 10-fold, preferably at least 15-fold, and preferably at least 20-fold, the number of normal cells (e.g., normal gastric cells) bound to the antibody.

[0025] Immunohistochemistry (IHC) may be used as a biological analysis method to evaluate antibody binding. It is considered preferable that tissue samples used for IHC are quickly frozen after excision, thawed, and fixed with acetone, as shown in Example 5, for example. Since CLDN18.2 is a tight junction protein found in normal tissues, positive staining of CLDN18.2 in normal and / or tumor tissues is mainly visualized as staining of membranous structures at cell-cell interfaces. Thus, negative or weak staining of CLDN18.2 means that membranous structures are not stained.

[0026] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 with a half maximal effective concentration (EC50) value of more than 0.4 μg / ml and less than 1 μg / ml, or an EC50 value of more than 0.5 μg / ml and less than 1 μg / ml, preferably an EC50 value of more than 0.6 μg / ml and less than 1 μg / ml, when measured by titration by flow cytometry (FC) using HEK293T cells overexpressing CLDN18.2. HEK293T cells overexpressing CLDN18.2 may be prepared by the method described in Example 3. The EC50 value of the antibody may be 0.4 to 1 μg / ml or 0.5 to 1 μg / ml, and is preferably 0.6 to 1 μg / ml, when measured by titration by flow cytometry (FC) using HEK293T cells overexpressing CLDN18.2.

[0027] Alternatively, when the EC50 value of the antibody is measured by flow cytometry using HEK293T cells overexpressing CLDN18.2, the EC50 value of the antibody may be compared with that of IMAB362, and the EC50 value of the antibody is at least 1.1 times, at least 1.2 times, preferably at least 1.5 times, more preferably at least 2 times, and even more preferably at least 2.5 times, and not more than 5 times, the EC50 value of IMAB362. When the EC50 value of the antibody is measured by flow cytometry using HEK293T cells overexpressing CLDN18.2, the EC50 value of IMAB362 is 1.1 to 2.5 times or 1.2 to 2.5 times, preferably 1.5 to 2.5 times, and more preferably 2 to 2.5 times.

[0028] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 with an EC50 value of more than 0.6 μg / ml and less than 3 μg / ml, or an EC50 value of more than 1 μg / ml and less than 3 μg / ml, preferably an EC50 value of more than 1.5 μg / ml and less than 3 μg / ml, more preferably an EC50 value of more than 2 μg / ml and less than 3 μg / ml, when measured by titration by flow cytometry (FC) using PA-TU-8988S-High cells. PA-TU-8988S-High cells may be produced by the method described in Example 2. The EC50 value of the antibody may be 0.6 to 3 μg / ml or 1 to 3 μg / ml, preferably 1.5 to 3 μg / ml, and more preferably 2 to 3 μg / ml, when measured by titration by flow cytometry (FC) using PA-TU-8988S-High cells.

[0029] Alternatively, when the EC50 value of the antibody is measured by flow cytometry using PA-TU-8988S-High cells, the EC50 value of the antibody may be compared with that of IMAB362, and the EC50 value of the antibody is at least 1.5 times, at least 2 times, preferably at least 3 times, more preferably at least 4 times, and not more than 5 times the EC50 value of IMAB362. The EC50 value of the antibody may be 1.5 to 5 times, 2 to 5 times, 3 to 5 times, or 4 to 5 times the EC50 value of IMAB362 when measured by flow cytometry (FC) using PA-TU-8988S-High cells.

[0030] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 with a maximum MFI value in the range of ±40% of the maximum MFI value of IMAB362, when binding to CLDN18.2 is measured by flow cytometry using HEK293T cells overexpressing CLDN18.2. The antibody or fragment thereof binds to CLDN18.2 with a maximum MFI value that is similar to or less than or equal to twice the maximum MFI value of IMAB362, when binding to CLDN18.2 is measured by flow cytometry using PA-TU-8988S-High cells.

[0031] Antibodies or functional fragments thereof that bind more strongly to tumor tissues expressing CLDN18.2 than to normal tissues expressing CLDN18.2 may have therapeutic advantages over antibodies that cannot distinguish between normal tissues expressing CLDN18.2 and tumor tissues expressing CLDN18.2. Tumor-specific antibodies are not expected to pose safety issues or side effects, which are very often related to the on-target effects of therapeutic antibodies on normal organs / tissues (Hansel et al., 2010). Such undesirable effects have been reported, for example, for IMAB362 (Sahin et al., 2018; Tureci et al., 2019).

[0032] The present invention provides an ADC comprising an antibody or a fragment thereof that binds to CLDN18.2 and a toxic drug, wherein the antibody or fragment thereof comprises, as heavy chain complementarity determining regions (HCDRs), an HCDR1 sequence of SEQ ID NO: 21, an HCDR2 sequence of SEQ ID NO: 22, and an HCDR3 sequence of SEQ ID NO: 23, and comprises, as light chain CDRs, an LCDR1 sequence of SEQ ID NO: 24, an LCDR2 sequence of SEQ ID NO: 25, and an LCDR3 sequence of SEQ ID NO: 26. In one embodiment, the toxic drug is an anthracycline.

[0033] In another embodiment, the present inventors have generated novel ADCs using the aforementioned novel anti-CLDN18.2 antibodies, which surprisingly showed better cytotoxic activity against tumor cells than a similar ADC using IMAB362.

[0034] The ADCs of the present invention have the general formula A-(LT): n having aA is an antibody or fragment thereof that binds to CLDN18.2, the antibody or fragment thereof comprising, as heavy chain complementarity determining regions (CDRs), the HCDR1 sequence of SEQ ID NO: 21, the HCDR2 sequence of SEQ ID NO: 22, and the HCDR3 sequence of SEQ ID NO: 23, and the light chain CDRs thereof comprising, as light chain CDRs, the LCDR1 sequence of SEQ ID NO: 24, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26; bL is a linker, cT is an anthracycline as a toxic drug.

[0035] In one embodiment, n is an integer from 1 to 10. The present invention further relates to pharma- ceutically acceptable salts or esters of the ADC.

[0036] Furthermore, the present invention provides an ADC comprising an antibody that binds to CLDN18.2, wherein the antibody comprises a heavy chain HCDR3 sequence of SEQ ID NO: 23 and a light chain LCDR3 sequence of SEQ ID NO: 26.

[0037] The consensus sequences for each CDR are shown in Table 1. The present invention encompasses any ADC that is based on any combination of CDRs derived from these consensus sequences and comprises an antibody or fragment thereof that binds to CLDN18.2. [Table 1]

[0038] In one embodiment, the linker L of the ADC of the invention comprises at least one non-cleavable linker element. A non-cleavable linker element is defined as a linker element that is only subject to lysosomal degradation, is not a substrate for specific enzymes, and is stable in plasma and the cytosol.

[0039] The non-cleavable linker element is Ethylenediamine (EDA), bN-formyl-N,N'-dimethylethylenediamine, c. diethylamine (DEA), d. The formula: [ka] Piperazine-derived compounds represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element, e. The formula: [ka] A compound represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element, f. The formula: [ka] A compound represented by the formula: (wherein the wavy line represents the bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof), g. The formula: [ka] A maleimidocaproyl compound represented by the formula: (wherein the wavy line indicates attachment to another linker element, and [Ab] indicates the antibody or fragment thereof), and h. The formula: [ka] A compound represented by the formula: (In the formula, the wavy line represents a bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof.) is selected from the group consisting of The non-cleavable linker element is linked to the toxic drug via an amide or ether bond.

[0040] The non-cleavable linker element may be directly covalently linked to the antibody (thereby forming a linker) or may be linked to the antibody via another linker element, such as an oligopeptide linker element. Additionally or alternatively, the linker may include a cleavable linker element.

[0041] The non-cleavable linker element may be linked to the antibody via an amino acid of the antibody sequence having a side chain with an available nucleophilic group, such as the ε-NH2 group of lysine or the sulfhydryl (SH) group of cysteine. Maleimide chemistry may be used to link to the side chain of cysteine, while acylation chemistry is typically used to link to the side chain of lysine. More information on such linkages is provided in Jain et al. (2015) (Jain et al., 2015). The linkage of the non-cleavable linker element to the oligopeptide linker element may be achieved by carbodiimide crosslinking chemistry. Guidelines for such crosslinking chemistries may be found in Thermo Scientific's Crosslinking Technical Handbook (2012) ("Crosslinking Technical Handbook", 2012).

[0042] The non-cleavable linker element may be directly linked to the anthracycline. In one embodiment, the non-cleavable linker element is 13 Amide bond at position C 14 The following formula (I): [ka] (wherein R1 is a hydrogen atom, a hydroxyl group or a methoxy group, and R2 is a C1-C5 alkoxy group). The anthracycline is linked to the anthracycline shown in

[0043] The linker may be formed using a combination of one or more linker elements, including an enzymatically cleavable linker element, to link the antibody and the toxic drug.

[0044] In another element, the linker further comprises an oligopeptide linker element, an enzymatically cleavable linker element, and / or a spacer element.

[0045] Said oligopeptide linker element is understood as an oligopeptide present in addition to the peptide chains forming said antibody or fragment thereof. Said oligopeptide linker element may be directly linked to the C-terminus of the heavy and / or light chains forming said antibody or fragment thereof. In one embodiment, the DNA sequence encoding said oligopeptide linker element may be part of the DNA encoding each heavy and / or each light chain forming said antibody or fragment thereof.

[0046] In another embodiment, the oligopeptide linker element may be generated by peptide ligation to link two or more oligopeptide linker elements. Ligation may be catalyzed by peptide ligases such as sortases (i.e., sortase A), asparaginyl endoproteases (i.e., butelase 1), trypsin-related enzymes (i.e., trypsiligase), and subtilisin-derived variants (i.e., peptiligase) (Nuijens et al., 2019). Thus, the oligopeptide linker element may include a peptide ligase recognition motif.

[0047] In the context of the present invention, a "spacer element" is understood as a spacer added to the linker to prevent steric hindrance and to allow proper attachment of a toxic drug to the antibody or fragment thereof.

[0048] In one embodiment, the oligopeptide linker element comprises the following as a sortase recognition motif: -LPXTG m -,-LPXAG m -,-LPXSG m -,-LAXTG m -,-LPXTG m -,-LPXTA m -, -NPQTG m - and - NPQTN m -, m is an oligoglycine, where m is an integer from 1 to 21;m is oligoalanine, where m is an integer from 1 to 21; m is oligoasparagine, where m is an integer from 1 to 21; and X is any conceivable amino acid. m is preferably 2 or 3, and particularly preferably 2. In a preferred embodiment, the oligopeptide as a sortase recognition motif is -LPQTGG- or -LPETGG-. The oligopeptide as a sortase recognition motif may be present at the C-terminus of the heavy chain and / or light chain of the antibody or fragment thereof, and is preferably present at the C-terminus of the light chain.

[0049] In a further preferred embodiment, the oligopeptide linker element of the ADC comprises the sequence of SEQ ID NO: 131. In one embodiment, the sequence of SEQ ID NO: 131 is present at the C-terminus of a heavy chain of the antibody, and in another preferred embodiment, is present at the C-terminus of a light chain of the antibody.

[0050] In another embodiment, the linker comprises an enzymatically cleavable linker element having the formula: [ka] (wherein the wavy line indicates the bond to another linker element and the bond to the antibody or toxic drug). The enzymatically cleavable linker element may be linked to another linker element or to the antibody or toxic drug using known cross-linking chemistries as described above for the non-cleavable linker element.

[0051] In yet another embodiment, the linker further comprises a spacer element. In one embodiment, the spacer element comprises a peptidic flexible oligopeptide. A flexible linker element can be used when some degree of flexibility or interaction is required for the linked components. Flexible oligopeptides are usually composed of small non-polar amino acids (e.g., G) or small polar amino acids (e.g., S or T). These small amino acids provide flexibility to the linked functional components, allowing them to move. The incorporation of S or T can form hydrogen bonds with water molecules to maintain the stability of the linker in aqueous solution, thereby suppressing undesirable interactions between the linker and protein moieties. For guidance on peptidic flexible oligopeptides, see Chen et al. (2013) (Chen, Zaro, and Shen, 2013).

[0052] The spacer element preferably comprises a peptidic flexible oligopeptide consisting of G and S, the peptidic flexible oligopeptide being (GGGGS) o (wherein o is 1, 2, 3, 4 or 5) is more preferred.

[0053] The present invention further provides an antibody-drug conjugate (ADC), comprising: a. The antibody-drug conjugate has the structure A-([oligopeptide linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [LPXTGG]-[ethylenediamine] and [ka] an antibody-drug conjugate selected from; b. The antibody-drug conjugate has the structure A-([oligopeptide linker element-enzyme-cleavable linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine] and ii. [LPXTGG]-[vc-PAB]-[piperazine] an antibody-drug conjugate selected from; c. The antibody-drug conjugate has the structure A-([spacer element-oligopeptide linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [GGGGS]-[LPXTGG]-[ethylenediamine] and [ka] an antibody-drug conjugate selected from: d. The antibody-drug conjugate has the structure A-([spacer element-oligopeptide linker element-enzyme-cleavable linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [GGGGS]-[LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine] and ii. [GGGGS]-[LPXTGG]-[vc-PAB]-[piperazine] The antibody-drug conjugate is selected from Here, A is an antibody or fragment thereof that binds to CLDN18.2, comprising an HCDR1 sequence of SEQ ID NO: 21, an HCDR2 sequence of SEQ ID NO: 22, and an HCDR3 sequence of SEQ ID NO: 23, and an LCDR1 sequence of SEQ ID NO: 24, an LCDR2 sequence of SEQ ID NO: 25, and an LCDR3 sequence of SEQ ID NO: 26, and T is an anthracycline.

[0054] In one embodiment, n is an integer from 1 to 10. The present invention further relates to pharma- ceutically acceptable salts or esters of the ADC.

[0055] The toxic drug may be linked via the linker to the C-terminus of the antibody heavy and / or light chain, or the C-terminus of the antibody fragment.

[0056] In a preferred embodiment, the non-cleavable linker element is ethylenediamine and the oligopeptide linker element is LPXTGG (where X is Q or E, preferably X is Q).

[0057] In one embodiment, a. (LT) is covalently linked to both light chains of said antibody; or b. (LT) is covalently linked to both heavy chains of said antibody; or c. (LT) is covalently linked to both light chains and both heavy chains of said antibody.

[0058] In one embodiment, a. (LT) is linked to the C-terminus of a light or heavy chain of the antibody; or b. (LT) is linked to an amino acid side chain of the light or heavy chain of said antibody.

[0059] When the toxic drug is linked to the C-terminus of the antibody light or heavy chain, the oligopeptide linker element and optional spacer element may be part of the amino acid sequence of the recombinantly expressed antibody with such a C-terminal tag, or when the toxic drug is linked to an amino acid side chain of the antibody amino acid sequence, the oligopeptide linker element may be attached using maleimide or acylation chemistry, depending on the amino acid side chain selected.

[0060] Surprisingly, ADCs of the invention in which the toxic drug is linked only to the heavy chain via an oligopeptide linker element-enzymatically non-cleavable linker element, or in which the toxic drug is linked only to the light chain via a spacer element-oligopeptide linker element-enzymatically non-cleavable linker element, or in which such linker-toxic drug combinations are linked to the heavy and light chains, had higher cytotoxic activity against CLDN18.2 expressing cells than a similar ADC using IMAB362 (see Figures 11-19 and Example 8), demonstrating that this newly identified antibody is also superior to prior art antibodies for use in ADCs. Furthermore, the ADCs of the invention have higher cytotoxic activity than an ADC using IMAB362 in which MMAE is linked via an MC-vc-PAB linker previously disclosed in WO2016 / 165762 (see Figure 11).

[0061] In one embodiment, the anthracycline has the following formula (I): [ka] (wherein R1 is a hydrogen atom, a hydroxyl group or a methoxy group, and R2 is a C1-C5 alkoxy group). In one embodiment, the anthracycline has a C 13 C 14 and the hydroxyl group is removed or the linker is 14 The hydroxyl group is removed by bonding at the hydroxyl position.

[0062] The toxic drug (C 13 Rank or C 14 Linking the antibody to the cytotoxic site does not affect the cytotoxic activity of the toxic drug.

[0063] For further information regarding the synthesis of PNU-159682 and its use as an anti-toxic drug in ADCs, reference may be made to the paper by Holte D et al. (2020) (Holte et al., 2020).

[0064] PNU-159682 may be linked to the antibody via a non-cleavable or an enzyme-cleavable linker, as shown below.

[0065] The linker may be a maleimide acetal linker. [ka]

[0066] Such linkers were used to generate an ADC consisting of PNU-159682-maleimide acetal-Ab, shown below. [ka]

[0067] Such an ADC consisting of PNU-159682-maleimide acetal-Ab is disclosed in U.S. Patent Publication No. 10,435,471, column 90. This NU-159682-maleimide acetal compound is disclosed in WO2010 / 009124 as compound 51, and may be prepared according to the description of Example 3d (paragraphs

[0576] to

[0578] ) using the compound prepared in Example 2 (paragraphs

[0542] to

[0550] ) of WO2010 / 009124.

[0068] PNU-159682 may be linked to the antibody via an enzyme-cleavable val-cit-PAB linker to generate an ADC consisting of PNU-159682-val-cit-PAB-Ab, as shown below. [ka]

[0069] Such ADCs are disclosed in U.S. Patent Publication No. 10,435,471 at columns 91-92. The PNU-159682-val-cit-PAB compound is disclosed in WO2010 / 009124 as compound 55 and may be prepared as described in Example 3b of WO2010 / 009124 (paragraphs

[0567] -

[0573] and FIG. 7d).

[0070] PNU-159682 may also be linked to the antibody via an enzyme-cleavable val-cit-PAB linker and an additional non-cleavable linker element, as shown below. [ka]

[0071] Such ADCs are disclosed in U.S. Patent Publication No. 10,435,471 at columns 91-92 and in Yu SF et al. Clin Cancer Research 2015 (Yu et al., 2015). Compounds consisting of PNU-159682-val-cit-PAB+non-cleavable linker may be prepared by the following procedure. [ka] Here, MC-val-cit-PAB is a commercially available product (MedChemExpress, catalog No.: HY-78738), and Boc is a tert-butyloxycarbonyl protecting group.

[0072] PNU-159682 may also be linked to the antibody via a non-cleavable maleimide linker as shown below. [ka]

[0073] Such an ADC is disclosed in U.S. Patent Publication No. 10,435,471, column 93. The PNU-159682-maleimide compound is disclosed in WO2010 / 009124 as compound 55, the preparation of which is disclosed in Example 3a (paragraphs

[0564] to

[0566] of said WO2010 / 009124).

[0074] Additionally, PNU-159682 has been linked to antibodies using a combination of non-cleavable, enzymatically cleavable and oligopeptide linker elements. Such an ADC is shown below. [ka]

[0075] Such compounds are disclosed in the article by Stefan et al. (Stefan et al., 2017). This ADC may be synthesized in a manner similar to the preparation of the ADC consisting of PNU-159682-val-cit-PAB+non-cleavable linker, except that Fmoc-Gly3-Val-Cit-PAB (commercial product of MedChemExpress, catalog number: HY-136106) is used instead of MC-Val-Cit-PAB, and the resulting compound consisting of the linker and the toxic drug may be linked to an antibody as described in the second paragraph on page 34 of WO2016 / 102679.

[0076] PNU-159682 may also be linked to the antibody via a combination of a non-cleavable EDA linker element and an oligopeptide linker element (-GGGGG-), as shown below. [ka]

[0077] Such a compound is disclosed in Figure 3A of WO2016 / 102679. The compound may be prepared according to the scheme in Figure 3B of WO2016 / 102679 and the description in the last paragraph on page 33 to the first paragraph on page 34, and the resulting compound consisting of a linker and a toxic drug may be linked to an antibody according to the description in the second paragraph on page 34 of WO2016 / 102679. The oligopeptide linker element used in the above preparation may be (-GGG-), and is preferably (-GG-).

[0078] The binding or binding affinity of an antibody is usually measured using the equilibrium binding constant (K a ) or the equilibrium dissociation constant (K d ) is the equilibrium binding constant (K a ) is the binding rate constant (k on ) and dissociation rate constant (k off ) and the equilibrium dissociation constant (K d ) is its reciprocal. Thus, the same affinity may be expressed in terms of different rate constants, as long as the ratio of the rate constants does not change. Binding affinity and / or rate constants can be measured using techniques known in the art or methods described herein, including ELISA, flow cytometric titration, isothermal titration calorimetry (ITC), Biacore (SPR), biolayer interferometry, fluorescence polarization, and the like. Depending on the characteristics of the antigen, the K a Value or K d In some cases, the K value is difficult to measure. This is especially true for integral membrane proteins such as claudins (Hashimoto et al., 2018). In such cases, the integral membrane protein may be expressed as a proteoliposome or lipoparticle. Such lipoparticles may also be immobilized on plastic and used in ELISA assays to measure the binding affinity of an antibody to an immobilized antigen. a Value or K dInstead of the ELISA value, the binding affinity (or binding strength) to an antigen may be examined by calculating the half effective concentration (EC50) value of each test antibody or its functional fragment. Example 2 and FIG. 1 described below show an example of a binding affinity curve by ELISA assay of an antibody having a CDR containing the consensus sequence shown in Table 1. The binding of an antibody to CLDN18.2 can be quantified using the EC50 value and the maximum binding value. Example 3 described below relates to the calculation of the EC50 value of an antibody having a CDR containing the consensus sequence shown in Table 1 by flow cytometry using cells expressing CLDN18.2.

[0079] The cytotoxic activity of an ADC can be evaluated by the EC50 value measured in an ADC cytotoxicity assay. Example 8 and Table 9 below relate to the calculation of the EC50 value of the ADC of the present invention by a cytotoxicity assay using CLDN18.2-expressing cells.

[0080] The EC50 values ​​(μg / ml) of binding of the hCl antibodies described herein were measured against the HEK293T or PA-TU-8988S-High cell lines overexpressing CLDN18.2, and were higher than the EC50 values ​​of the reference antibody IMAB362 against the same cell lines for all hCl antibodies (see Table 4 and Example 2), i.e., the binding of the hCl antibodies described herein to CLDN18.2 was lower affinity than IMAB362, but surprisingly the EC50 values ​​(ng / ml) of ADC cytotoxicity of the ADCs of the invention measured against the HEK293T or A549 cell lines overexpressing CLDN18.2, or the PA-TU-8988S-High cell line, were lower than the EC50 values ​​of ADC cytotoxicity using IMAB362 measured against the same cell lines (see Table 9 and Example 8). These results demonstrated that, although the binding affinity of the hCl antibody to the target is lower than that of IMAB362, the ADC of the present invention has higher cytotoxic activity than the ADC using IMAB362.

[0081] Similarly, the ADCs of the invention were shown to be more effective in vivo in patient-derived tumor xenograft models than the ADCs with IMAB362 (see Example 9).

[0082] In one embodiment, the antibody or fragment thereof binds to CLDN18.2 and comprises, as heavy chain CDRs, the HCDR1 sequence of SEQ ID NO: 21, the HCDR2 sequence of SEQ ID NO: 126, and the HCDR3 sequence of SEQ ID NO: 23, and, as light chain CDRs, the LCDR1 sequence of SEQ ID NO: 24, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26.

[0083] In one embodiment, the antibody or fragment thereof binds to CLDN18.2, a. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 15 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; b. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; c. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16, and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:17, the LCDR2 sequence of SEQ ID NO:14, and the LCDR3 sequence of SEQ ID NO:11; d. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:18, the LCDR2 sequence of SEQ ID NO:19 and the LCDR3 sequence of SEQ ID NO:11; e. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 15 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; f. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:20 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; g. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:20, and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:18, the LCDR2 sequence of SEQ ID NO:19, and the LCDR3 sequence of SEQ ID NO:11; h. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 20 and the HCDR3 sequence of SEQ ID NO: 8, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; or i. HCDR1 sequence of SEQ ID NO: 12, HCDR2 sequence of SEQ ID NO: 20, and HCDR3 sequence of SEQ ID NO: 8, and LCDR1 sequence of SEQ ID NO: 17, LCDR2 sequence of SEQ ID NO: 14, and LCDR3 sequence of SEQ ID NO: 11 Includes.

[0084] In another preferred embodiment, the ADC using the antibody has a higher cytotoxic activity against CLDN18.2-expressing cells than a similar ADC using IMAB362, as shown, for example, by the EC50 value for cytotoxic activity.

[0085] In yet another embodiment, the antibody or fragment thereof binds to CLDN18.2 and a. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:2 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; b. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:7 and the HCDR3 sequence of SEQ ID NO:8, and the LCDR1 sequence of SEQ ID NO:9, the LCDR2 sequence of SEQ ID NO:10 and the LCDR3 sequence of SEQ ID NO:11; or c. The HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 2, and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 13, the LCDR2 sequence of SEQ ID NO: 14, and the LCDR3 sequence of SEQ ID NO: 11 Includes.

[0086] In yet another embodiment, the antibody or fragment thereof binds to CLDN18.2 and a. The VH sequence of SEQ ID NO:27 and the VL sequence of SEQ ID NO:28; b. A VH sequence of SEQ ID NO:29 and a VL sequence of SEQ ID NO:30; or c. The VH sequence of SEQ ID NO:31 and the VL sequence of SEQ ID NO:32 Includes.

[0087] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 and a. The VH sequence of SEQ ID NO: 33; b. The VH sequence of SEQ ID NO:34; c. The VH sequence of SEQ ID NO:35; d. The VH sequence of SEQ ID NO: 36; or e. The VH sequence of SEQ ID NO: 37; f. The VL sequence of SEQ ID NO:38; g. The VL sequence of SEQ ID NO:39; h. the VL sequence of SEQ ID NO: 40; or i. The VL sequence of SEQ ID NO: 41 Includes:

[0088] In yet another embodiment, the antibody or fragment thereof binds to CLDN18.2 and a. The VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 38; b. the VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:38; c. A VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:39; d. The VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:40; e. the VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:38; f. the VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:41; g. The VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:40; h. the VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:41; i. the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38; or j. The VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:39 Includes.

[0089] In another embodiment, the antibody binds to CLDN18.2 a. The heavy chain sequence of SEQ ID NO:46 and the light chain sequence of SEQ ID NO:51; b. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:51; c. A heavy chain sequence of SEQ ID NO:47 and a light chain sequence of SEQ ID NO:52; d. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:53; e. a heavy chain sequence of SEQ ID NO:48 and a light chain sequence of SEQ ID NO:51; f. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:54; g. A heavy chain sequence of SEQ ID NO:49 and a light chain sequence of SEQ ID NO:53; h. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:54; i. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:51; or j. The heavy chain sequence of SEQ ID NO:50 and the light chain sequence of SEQ ID NO:52 Includes.

[0090] The light chain constant region (CL) of the antibody disclosed herein may have the amino acid sequence of SEQ ID NO: 127, and the heavy chain constant region CH1 and Fc region may have the amino acid sequence of SEQ ID NO: 128.

[0091] The ADCs of the present invention in which an anthracycline is bound only to the light chain have higher cytotoxic activity against CLDN18.2-expressing cells than IMAB362 in which an anthracycline derivative is bound only to the light chain (see Figures 11 to 19). Furthermore, the ADCs of the present invention in which an anthracycline is bound to both the heavy and light chains, only the heavy chain, or only the light chain have higher cytotoxic activity than IMAB362-MC-vc-PAB-MMAE previously disclosed in WO2016 / 165762 (see Figure 11).

[0092] Additionally, the ADCs of the invention have been shown to have greater in vivo cytotoxic activity against patient-derived gastric, colon, pancreatic and lung tumor xenograft models than an identical ADC using IMAB362 (see Figures 21-24 and Example 9, respectively). In a preferred embodiment, the antibody binds to CLDN18.2 and comprises a heavy chain sequence of SEQ ID NO:46 and a light chain sequence of SEQ ID NO:51.

[0093] In a further preferred embodiment, the antibody binds to CLDN18.2 and consists of a heavy chain sequence of SEQ ID NO:46 and a light chain sequence of SEQ ID NO:51.

[0094] The antibody may bind more strongly to tumor cells expressing CLDN18.2 than to normal gastric cells expressing CLDN18.2, and may have an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to the amino acid sequence of an antibody of the present invention.

[0095] In one embodiment, the antibody binds to CLDN18.2 a. The VH sequence of SEQ ID NO:27 and the VL sequence of SEQ ID NO:28; b. A VH sequence of SEQ ID NO:29 and a VL sequence of SEQ ID NO:30; or c. The VH sequence of SEQ ID NO:31 and the VL sequence of SEQ ID NO:32 The antibody has an amino acid sequence that has at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 98% identity to an antibody comprising the amino acid sequence of

[0096] In yet another embodiment, the antibody binds to CLDN18.2 and a. The VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 38; b. the VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:38; c. A VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:39; d. The VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:40; e. the VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:38; f. the VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:41; g. The VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:40; h. the VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:41; i. the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38; or j. The VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:39 The antibody has an amino acid sequence that has at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 98% identity to an antibody comprising the amino acid sequence of

[0097] In yet another embodiment, the antibody binds to CLDN18.2 and has an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 98% identity to an antibody consisting of a heavy chain sequence of SEQ ID NO: 46 and a light chain sequence of SEQ ID NO: 51.

[0098] In another embodiment, the Fc domain of the antibody (or antibody fragment, if present) may comprise modifications or mutations, such as those listed in Table 2 below. Such modifications or mutations may be introduced to modulate the effector activity of the Fc domain of the antibody. Antibody modifications may further include peptide tags added to the C-terminus of the antibody heavy and / or light chains. Such tags may be used, for example, for protein purification or protein labeling. In another embodiment, the antibody or fragment thereof that binds to CLDN18.2 is in the form of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, Fv, scFv, IgGACH2, F(ab')2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, non-depleting IgG (IgG that does not induce cell loss), diabody, bivalent antibody, or Fc recombinant form thereof. In a preferred embodiment, the antibody is an IgG1 antibody. The Fc region of an immunoglobulin interacts with various types of Fcγ receptors (FcγR) and complement proteins (e.g., C1q) and is responsible for the effector functions of the immune system (e.g., elimination of target cells) through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). Enhancing or suppressing the effector functions associated with the Fc region may be beneficial for therapeutic applications. The type of immunoglobulin (IgA, IgD, IgE, IgG, IgM) may be selected depending on the desired effector function of the antibody associated with the Fc domain. Synthetic immunoglobulins may also be used, such as immunoglobulins containing amino acid residues 118-260 of IgG2 and amino acid residues 261-447 of IgG4, or IgG2 variants with point mutations (e.g., H268Q / V309L / A30S / P331S) derived from IgG4. Such synthetic immunoglobulins have reduced antibody effector functions. Furthermore, in order to regulate the effector functions of antibodies, immunoglobulins having modified Fc regions may be used.An example of such a genetic modification of the Fc region is shown in Table 2. Additionally, fucosylated carbohydrate chains may be expressed from recombinant protein-producing cell lines to affect binding to Fcγ receptors. [Table 2]

[0099] The half-life of the antibody in vivo may be modulated. The Fc domain plays a central role in the stability and serum half-life of the antibody. In therapeutic applications, the half-life of the antibody may be shortened by using antibody fragments with the Fc domain removed or truncated, such as F(ab)2, Fv, scFv, IgGACH2, F(ab')2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, etc. The antibody may be in the form of a diabody or bivalent antibody. The use of a diabody or bivalent antibody may increase the affinity to the target and allow for lower doses. The functional fragments with the Fc domain removed or truncated may be used to develop alternative therapeutic methods, such as chimeric antigen receptor T cells (CAR T cells) and bispecific T cell-inducing antibodies (BiTE). In a CAR construct, one VH domain and one VL domain are usually linked via a short peptide linker to form a single chain variable fragment (scFv), which is further linked to a transmembrane domain and a cytoplasmic T cell immunoreceptor activation tyrosine motif (e.g., from CD3ζ), which is further linked to a costimulatory molecule domain (e.g., from CD28, 4-1BB (CD127) or OX40) (Chang and Chen, 2017). The VH and VL domains used in the scFv fragment may be any of the antibodies listed in Table 3. BiTEs are usually fused together with two scFvs from two different antibodies. One scFv domain may be derived from any of the isolated antibodies that bind to CLDN18.2 listed in Table 3, and the other scFv domain may be derived from an antibody that binds, for example, CD3, CD16, NKG2D, NKp46, CD2, CD28, or CD25.For detailed guidance on the BiTE antibody format and other bispecific antibody formats used for T cell targeting, please see the review article by Diego Ellerman (2019).

[0100] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 and has a light chain constant region (CL) of SEQ ID NO: 127, and preferably further has a heavy chain constant region CH1 domain and Fc region of SEQ ID NO: 129, which has L234A / L235A mutations in the CH2 domain of the heavy chain constant region, thereby reducing FcγR binding. More preferably, the antibody has a heavy chain constant region CH1 domain and Fc region of SEQ ID NO: 130, which has L234A / L235A / P329G mutations in the CH1 domain and Fc region of the heavy chain constant region, thereby further reducing FcγR binding.

[0101] Surprisingly, it was shown herein that an ADC of the present invention using an antibody with L234A / L235A mutations in the CH2 domain of the heavy chain constant region had higher cytotoxic activity in vivo against patient-derived tumor xenograft models than an otherwise identical ADC using IMAB362 (see Figures 21C and 23B and Example 9).

[0102] In another preferred embodiment, the antibody or fragment thereof binds to CLDN18.2 and comprises a VH sequence of SEQ ID NO: 33, a VL sequence of SEQ ID NO: 38, a light chain constant region (CL) of SEQ ID NO: 127, and a heavy chain constant region CH1 and Fc region of SEQ ID NO: 129 with L234A / L235A mutations.

[0103] In another preferred embodiment, the antibody or fragment thereof binds to CLDN18.2 and consists of a VH sequence of SEQ ID NO: 33, a VL sequence of SEQ ID NO: 38, a light chain constant region (CL) of SEQ ID NO: 127, and a heavy chain constant region CH1 and Fc region of SEQ ID NO: 129 with L234A / L235A mutations.

[0104] In another embodiment, the antibody or fragment thereof binds to CLDN18.2 and is humanized.The humanization of monoclonal antibodies has already been well established.Handbook of Therapeutic Antibodies, Second Edition provides sufficient information on the humanization of monoclonal antibodies (Saldanha, 2014), bioinformatics tools for analyzing such antibodies (Martin and Allemn, 2014), and the development and production of therapeutic antibodies (Jacobi et al., 2014).

[0105] In another embodiment, the antibody or fragment thereof is an isolated antibody or isolated fragment that binds to CLDN18.2.

[0106] In yet another embodiment, the antibody or fragment thereof binds to CLDN18.2 but not to CLDN18.1. Thus, the antibody does not show cross-reactivity or cross-binding to CLDN18.1. The binding of the antibody to the target protein can be tested by performing flow cytometry on cells expressing the target protein. The specific binding of the tested antibody to the target protein can be visualized in a histogram plot. If the tested antibody specifically binds to the expressed target protein, a high fluorescent signal peak is observed in the histogram plot, and if the tested antibody does not bind or can only bind very weakly to the expressed target protein, a low fluorescent signal peak is observed in the histogram plot. The degree of binding can also be shown as a bar graph showing the maximum mean fluorescence intensity (maximum MFI value) measured by flow cytometry, where a high maximum MFI value indicates strong binding and a low or no maximum MFI value indicates very weak binding or no binding. The affinity of the antibody to the target may be examined by comparing the maximum MFI values ​​of various antibodies in the same experimental setup, the higher the maximum MFI value, the less likely it is to dissociate and the higher the affinity. An example of such a binding assay is shown in Example 3 and Figures 4 and 5.

[0107] In another embodiment, the ADC is linked to another moiety. The antibody or its fragment may be linked to another moiety by covalent or non-covalent bond. The other moiety may include a radioisotope, a fluorescent tag, a histological marker, a cytotoxin or a cytokine. The other moiety may be linked to the antibody by a linker known in the art.

[0108] In yet another embodiment, the specific antibody or fragment thereof binds to CLDN18.2 and is less susceptible to post-translational deamidation than IMAB362. In yet another embodiment, the tumor-specific antibody or fragment thereof binds to CLDN18.2 and is less susceptible to post-translational deamidation. Post-translational modifications (PTMs) are an important concern in antibody development and antibody manufacturing and storage. Unregulated post-translational modifications may compromise the efficacy, activity, potency or stability of antibodies. Post-translational modifications may be N-glycosylation, glycosylation of lysine, capping of cysteines with additional cysteines from cell culture media during bioprocessing, glutathione or other sulfhydryl-containing compounds, or formation of dimers or higher oligomers via cysteines linked by covalent disulfide bridges. Among post-translational modifications, deamidation of asparagine (Asn, N) residues, isomerization of aspartate (aspartic acid, Asp, D) residues, and formation of succinimide intermediates are the most frequent in vivo modification reactions following the manufacture, storage, or administration of therapeutic antibodies. Asparagine deamidation and aspartic acid isomerization depend on sequence fidelity, structural environment, and storage conditions (particularly solution pH and storage temperature). These modifications can result in reduced or even loss of function or biological activity, especially when the affected residues are involved in target binding. Asparagine and aspartic acid residues are at risk of modification, especially when they are present in structurally flexible regions such as CDR loops, or when certain other structural requirements are met. On the other hand, framework regions have been observed to be relatively resistant to modification. In addition to the structural locations of asparagine and aspartic acid residues, typical motifs in which asparagine deamidation or aspartic acid isomerization occurs have been identified.Typical motifs in which asparagine deamidation occurs include NG, NS, NN, NT, and NH, and typical motifs in which aspartic acid isomerization occurs include DG, DS, DD, DT, and DH (Lu et al., 2019). In silico analysis has revealed that the antibodies disclosed herein have a DG motif in which aspartic acid isomerization can occur in the last amino acid of CDR2 of the VL domain and in the CH2 and CH3 regions of the heavy chain (VL-CDR2 (position 62), CH2 (position 282), and CH3 (position 403)).

[0109] Aspartic acid isomerization can be assessed by exposing the antibody to low pH (i.e., pH 5.5) and heat (i.e., 40°C) for two weeks, while asparagine deamidation of the antibody can be assessed by exposing the antibody to high pH (i.e., pH 8.0) and heat (i.e., 40°C) for one week, mimicking manufacturing and storage conditions.

[0110] Herein, the inventors have demonstrated that, although the antibodies disclosed herein contain asparagine and aspartic acid in the CDR and have an aspartic acid-glycine (DG) motif in which isomerization of aspartic acid can occur, they surprisingly did not undergo deamidation of asparagine (see Table 6) or isomerization of aspartic acid (see Table 7) even under the harsh conditions described above, and no effect was observed on the binding affinity to CLDN18.2. On the other hand, in IMAB362, deamidation of asparagine was observed under the harsh conditions described above, and the binding affinity was reduced (see Table 6 and FIG. 10). Thus, the present invention provides an isolated antibody or a fragment thereof that binds to CLDN18.2, which is less susceptible to post-translational modification than IMAB362 during production, storage, and (in vivo) clinical application, and which reliably maintains its binding affinity to CLDN18.2 during production, storage, and (in vivo) clinical application.

[0111] In one embodiment, the antibody binds to the same epitope as an antibody comprising the heavy chain sequence of SEQ ID NO:46 and the light chain sequence of SEQ ID NO:51.

[0112] The invention further provides an antibody that competes for binding with an antibody described herein, hi one embodiment, the antibody competes for binding with an antibody comprising the heavy chain sequence of SEQ ID NO: 46 and the light chain sequence of SEQ ID NO: 51.

[0113] The present invention further provides an antibody that competitively inhibits the binding of an antibody described herein to claudin 18.2. In one embodiment, the antibody competitively inhibits the binding of an antibody comprising the heavy chain sequence of SEQ ID NO: 46 and the light chain sequence of SEQ ID NO: 51 to claudin 18.2.

[0114] A suitable method for detecting the binding of multiple antibodies to the same antigen is the antigen-antibody interaction mapping method, which has been reported by Abbott (2014) (Abbott, Damschroder, & Lowe, 2014). A suitable method for detecting competition is the epitope binning competition assay, which has been reported by Abdiche (2009) (Abdiche et al., 2009). Also, a suitable method for detecting competitive inhibition is the ELISA assay.

[0115] In another embodiment, the invention relates to a method of producing an ADC of the invention.

[0116] In one embodiment, the method further comprises: a. providing as A an antibody or fragment thereof having one or more linker elements; b. providing as T one or more toxic drugs having one or more linker elements; and c. Linking the antibody and the toxic drug to obtain an antibody-drug conjugate. Includes.

[0117] In one embodiment, the method further comprises: d. providing as A an antibody or fragment thereof having an oligopeptide linker element in its light and / or heavy chain, preferably at its C-terminus, and optionally a spacer element between said light and / or heavy chain and said oligopeptide linker element; e. providing as T one or more toxic drugs having a non-cleavable linker element to which an oligopeptide linker element may be attached; and f. Linking the antibody and the toxic drug to obtain an antibody-drug conjugate. Includes.

[0118] Antibody A disclosed herein may be attached with any oligopeptide linker element disclosed herein, and may be attached with any spacer element as an optional component. Similarly, toxic drug T, an anthracycline, may be linked with any non-cleavable linker element disclosed herein. The type of linkage may depend on the linker element and / or the method of preparation of the ADC. A diagram of an ADC produced by the method is shown in FIG. 25.

[0119] In one preferred embodiment, the ADC of the invention comprises: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 46 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. It consists of:

[0120] In another preferred embodiment, the ADC of the invention comprises: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 133 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. It consists of:

[0121] In yet another preferred embodiment, the ADC of the invention comprises: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 134 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. It consists of:

[0122] In addition, the invention relates to pharmaceutical compositions comprising the ADCs disclosed herein and an excipient.

[0123] The present invention further provides a nucleic acid sequence encoding an isolated tumor-specific antibody or functional fragment thereof that binds to CLDN18.2 for use in the manufacture of ADCs. The nucleic acid sequence may encode only the CDRs of the antibody, may encode the variable heavy (VH) and variable light (VL) regions of the antibody, or may encode the full-length heavy and light chains of the antibody. Such nucleic acid sequences are shown in Table 3. The nucleic acid sequence may encode F(ab)2, Fv, scFv, IgGACH2, F(ab')2, scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, non-depleting IgG, diabody, bivalent antibody, or Fc recombinants thereof. The immunoglobulin encoded by the nucleic acid sequence may be IgA1, IgA2, IgD, IgE, IgG1, IdG2, IgG3, IgG4, synthetic IgG, IgM, or variants or Fc recombinants thereof. The nucleic acid may further comprise a coding sequence for an oligopeptide linker element fused directly to the C-terminus of the heavy and / or light chain of the antibody.

[0124] Furthermore, the present invention provides an expression vector comprising the nucleic acid of the present invention or a degenerate nucleic acid resulting from codon degeneracy. The expression vector may be an expression vector for expressing a protein in mammalian cells, bacterial cells, fungal cells or insect cells, and may be selected depending on the type of host cell into which the expression vector comprising the nucleic acid encoding the antibody or functional fragment thereof is introduced. Detailed guidance on the construction of such vectors is provided in the book by Green and Sambrook (Green and Sambrook, 2012). Expression vectors for mammalian cells are preferred, and expression vectors for CHO cells are particularly preferred.

[0125] The present invention further provides a host cell comprising a nucleic acid or expression vector of the invention. The host cell may be a mammalian cell or mammalian cell line, a bacterial cell, a fungal cell or an insect cell. Mammalian cells are preferred, CHO cells being particularly preferred.

[0126] In another embodiment, the invention relates to an ADC of the invention that binds CLDN18.2 for use in therapy.

[0127] In another embodiment, the invention relates to an ADC of the invention for use in treating a subject suffering from a cancer / neoplastic disease.

[0128] In another embodiment, the invention relates to an ADC for use in treating a subject at risk of developing and / or diagnosed with a neoplastic disease.

[0129] The ADCs disclosed herein may be used as monotherapy. In a preferred embodiment, the ADCs disclosed herein are used in combination with an established standard of care treatment for a neoplastic disease.

[0130] The neoplastic disease may be at least one selected from the group consisting of pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer and lung cancer. The neoplastic disease to be treated expresses CLDN18.2.

[0131] In one embodiment, the subject is a mammal. In a preferred embodiment, the subject is a human.

[0132] Another embodiment of the invention provides a method of treating a neoplastic disease, such as pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, lung cancer, etc., using an ADC provided herein, comprising administering to a subject in need of treatment for the neoplastic disease a pharmacologic effective amount of said ADC. The treatment method may be a monotherapy, but is preferably a combination therapy in combination with an established standard of care for the neoplastic disease.

[0133] The amino acid sequence of human CLDN18.2 protein has the NCBI reference sequence NP_001002026.1. This sequence may be derived from SEQ ID NO: 135. [Brief description of the drawings]

[0134] [Figure 1] Binding of selected chimeric or humanized anti-CLDN18.2 antibodies as indicated in the graph to CLDN18.2-containing or null lipoparticles was assessed by ELISA. A. Chimeric antibodies cCl1-1, cCl1-2 and cCl1-3, IMAB362 and secondary antibody only; B. Humanized antibodies hCl1a-hCl1j, chimeric antibodies cCl1-1, IMAB362 and secondary antibody only. All newly generated antibodies bind to CLDN18.2 on liposomes.

[0135] [Diagram 2] Sorting of PA-TU-8988S cells based on the expression level of CLDN18.2. A. Flow cytometry profile of PA-TU-8988S cells stained with IMAB362. B. Flow cytometry profile of PA-TU-8988S cells sorted by FACS based on high expression of CLDN18.2.

[0136] [Diagram 3]Generation of HEK293T cells overexpressing huCLDN18.2. HEK293T cells do not endogenously express CLDN18.2 and were transfected with a plasmid encoding huCLDN18.2 to stably express CLDN18.2 or with a plasmid encoding huCLDN18.1 to stably express CLDN18.1. Expression was analyzed by flow cytometry after staining with IMAB362, pan-CLDN18.1 antibody or anti-human IgG secondary antibody alone. A. Flow cytometry profile of untransfected HEK293T cells. B. Flow cytometry profile of transfected HEK293T cells stably expressing CLDN18.1. C. Flow cytometry profile of transfected HEK293T cells stably expressing CLDN18.2.

[0137] [Figure 4] Flow cytometry assay analysis of the binding of chimeric cCl1-1, cCl1-2, or cCl1-3 antibodies to pre-B cell L11 cell lines overexpressing CLDN18.1 or CLDN18.2. These chimeric antibodies bind to CLDN18.2 but not to CLDN18.1. IMAB362 was used as a control showing positive binding.

[0138] [Diagram 5] Flow cytometry assay of binding of humanized antibodies hCl1a-hCl1j to HEK293T cells overexpressing CLDN18.1 or CLDN18.2. These humanized antibodies bind to CLDN18.2 but not to CLDN18.1. IMAB362 and cCl1-1 were used as positive binding controls.

[0139] [Figure 6]1 shows the FACS expression profile of A549 cells overexpressing CLDN18.2. A549 cells do not endogenously express CLDN18.2, but were stably transfected with a plasmid encoding CLDN18.2 and analyzed for CLDN18.2 expression by FACS using IMAB362.

[0140] [Figure 7] 1 shows live cell staining by flow cytometry. Graph showing percentage of isolated single cells bound by CLDN18.2 antibodies (cCl1-1, hCl1a, hCl1b, hCl1c, hCl1f or IMAB362). The single cells were isolated from CLDN18.2-expressing mouse tumors grown from injected CLDN18.2-overexpressing A549 cells (black bars) or from mouse normal stomachs expressing CLDN18.2 (white bars).

[0141] [Figure 8] Staining of frozen stomach tissue is shown. Frozen tissue slides of normal mouse stomach tissue expressing CLDN18.2 stained with hCl1a antibody (A), hCl1b antibody (B), hCl1c antibody (C), hCl1f antibody (D), or IMAB362 antibody (E) are shown. The photographs show representative IHC staining images.

[0142] [Figure 9] Shown are staining of frozen sections of tumor tissues grown from injected CLDN18.2-overexpressing A549 cells. Frozen tissue slides of mouse tumors expressing CLDN18.2 stained with hCl1a (A), hCl1f (B), IMAB362 (C) or Abcam's pan-CLDN18 antibody [34H14L15]. Photographs show representative IHC staining images.

[0143] [Figure 10] 1 shows the effect of deamidation on the binding activity of IMAB362. Deamidation reduces the affinity of IMAB362 for CLDN18.2.

[0144] [Figure 11] The results of in vitro cytotoxicity assays against HEK-293T-CLDN18.2 cells were performed using ADCs of chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C) in which only the heavy chain, only the light chain, or both the heavy and light chains were conjugated with PNU. The cytotoxic activity of each ADC was compared to that of an ADC in which IMAB362 and PNU were conjugated in the same manner, and an ADC in which the isotype control Ac10 and PNU were conjugated in the same manner. The results were also compared to an ADC in which IMAB362 was conjugated to the toxic drug MMAE via the enzyme-cleavable linker MC-vc-PAB, as shown in the figure. Legend in the figure: ADCs in which PNU was conjugated to the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs in which PNU was conjugated to only the heavy chain are denoted as HC-PNU, and ADCs in which PNU was conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugates contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is conjugated to the light chain only, the flexible oligopeptide -GGGGS- is also included. The G2 in the notation represents two glycines in the oligopeptide linker.

[0145] [Figure 12]The results of an in vitro cytotoxicity assay against HEK-293T-CLDN18.1 cells were performed using ADCs with PNU conjugated to only the heavy chain, only the light chain, or both the heavy and light chains of the chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C). The cytotoxic activity of each ADC was compared to that of an ADC with IMAB362 and PNU conjugated in the same manner, and an ADC with the isotype control Ac10 and PNU conjugated in the same manner. Legend in the figure: ADCs with PNU conjugated to the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs with PNU conjugated to only the heavy chain are denoted as HC-PNU, and ADCs with PNU conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugation contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is attached to the light chain alone, the flexible oligopeptide -GGGGS- is also included, where G2 represents two glycines in the oligopeptide linker.

[0146] [Figure 13] The results of an in vitro cytotoxicity assay against BxPC-3-CLDN18.2 cells using ADCs with PNU conjugated to only the heavy chain, only the light chain, or both the heavy and light chains of the chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C) are shown. The cytotoxic activity of each ADC was compared to that of an ADC with IMAB362 and PNU conjugated in the same manner, and an ADC with the isotype control Ac10 and PNU conjugated in the same manner. Legend in the figure: ADCs with PNU conjugated to the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs with PNU conjugated to only the heavy chain are denoted as HC-PNU, and ADCs with PNU conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugation contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is attached to the light chain alone, the flexible oligopeptide -GGGGS- is also included, where G2 represents two glycines in the oligopeptide linker.

[0147] [Figure 14] The results of in vitro cytotoxicity assays against A549-CLDN18.2 cells using ADCs with PNU conjugated to only the heavy chain, only the light chain, or both the heavy and light chains of chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C). The cytotoxic activity of each ADC was compared to that of an ADC with IMAB362 and PNU conjugated in the same manner, and an ADC with the isotype control Ac10 and PNU conjugated in the same manner. The results were also compared to an ADC with IMAB362 and the toxic drug MMAE conjugated via an enzyme-cleavable linker, MC-vc-PAB. Legend in the figure: ADCs with PNU conjugated to both the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs with PNU conjugated to only the heavy chain are denoted as HC-PNU, and ADCs with PNU conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugates contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is conjugated to the light chain only, the flexible oligopeptide -GGGGS- is also included. The G2 in the notation represents two glycines in the oligopeptide linker.

[0148] [Figure 15]The results of in vitro cytotoxicity assays against A549-CLDN18.1 cells using ADCs with PNU conjugated to only the heavy chain, only the light chain, or both the heavy and light chains of chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C). The cytotoxic activity of each ADC was compared to that of an ADC with IMAB362 and PNU conjugated in the same manner, and an ADC with the isotype control Ac10 and PNU conjugated in the same manner. The results were also compared to an ADC with IMAB362 and the toxic drug MMAE conjugated via an enzyme-cleavable linker, MC-vc-PAB. Legend: ADCs with PNU conjugated to both the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs with PNU conjugated to only the heavy chain are denoted as HC-PNU, and ADCs with PNU conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugates contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is conjugated to the light chain only, the flexible oligopeptide -GGGGS- is also included. The G2 in the notation represents two glycines in the oligopeptide linker.

[0149] [Figure 16]The results of an in vitro cytotoxicity assay against PATU-8988-S-High cells were performed using ADCs with PNU conjugated to only the heavy chain, only the light chain, or both the heavy and light chains of the chimeric antibodies cCl1-1 (A), cCl1-2 (B), or cCl1-3 (C). The cytotoxic activity of each ADC was compared to that of an ADC with IMAB362 and PNU conjugated in the same manner, and an ADC with the isotype control Ac10 and PNU conjugated in the same manner. Legend in the figure: ADCs with PNU conjugated to the heavy and light chains of the antibody are denoted as HC-LC-PNU, ADCs with PNU conjugated to only the heavy chain are denoted as HC-PNU, and ADCs with PNU conjugated to only the light chain are denoted as LC-PNU. All ADCs containing PNU conjugation contain the oligopeptide linker -LPQTGG- and an ethylenediamine linker. When PNU is attached to the light chain alone, the flexible oligopeptide -GGGGS- is also included, where G2 represents two glycines in the oligopeptide linker.

[0150] [Figure 17] The results of an in vitro cytotoxicity assay against A549-CLDN18.2 cells using ADCs in which PNU was conjugated only to the light chain of humanized antibodies hCl1a-hCl1c (A), hCl1d-hCl1f (B), hCl1g-hCl1i (C), or hCl1j (D) are shown. The cytotoxic activity of each ADC was compared with the cytotoxic activity of ADCs in which PNU was conjugated only to the light chain of the chimeric cCl1-1 antibody or IMAB362. Legend in the figure: ADCs labeled LC-PNU are ADCs in which PNU is conjugated only to the light chain via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker. G2 in the notation represents two glycines in the oligopeptide linker.

[0151] [Figure 18]The results of an in vitro cytotoxicity assay against HEK-293T-CLDN18.2 cells using ADCs in which PNU was conjugated only to the light chain of humanized antibodies hCl1a-hCl1c (A), hCl1d-hCl1f (B), hCl1g-hCl1i (C), or hCl1j (D) were used. The cytotoxic activity of each ADC was compared with that of ADCs in which PNU was conjugated only to the light chain of the chimeric cCl1-1 antibody or IMAB362. Legend in the figure: ADCs labeled LC-PNU are ADCs in which PNU was conjugated only to the light chain via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker. G2 in the notation represents two glycines in the oligopeptide linker.

[0152] [Figure 19] The results of an in vitro cytotoxicity assay against HEK-293T-CLDN18.1 cells using ADCs in which PNU was conjugated only to the light chain of humanized antibodies hCl1a-hCl1c (A), hCl1d-hCl1f (B), hCl1g-hCl1i (C), or hCl1j (D) are shown. The cytotoxic activity of each ADC was compared with that of ADCs in which PNU was conjugated only to the light chain of the chimeric cCl1-1 antibody or IMAB362. Legend in the figure: ADCs labeled LC-PNU are ADCs in which PNU was conjugated only to the light chain via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker. G2 in the notation represents two glycines in the oligopeptide linker.

[0153] [Figure 20]The results of an in vitro cytotoxicity assay against PATU-8988-S-High cells using ADCs in which PNU was conjugated only to the light chain of humanized antibodies hCl1a-hCl1c (A), hCl1d-hCl1f (B), hCl1g-hCl1i (C), or hCl1j (D) are shown. The cytotoxic activity of each ADC was compared with the cytotoxic activity of ADCs in which PNU was conjugated only to the light chain of the chimeric cCl1-1 antibody or IMAB362. Legend in the figure: ADCs labeled LC-PNU are ADCs in which PNU was conjugated only to the light chain via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker. G2 in the notation represents two glycines in the oligopeptide linker.

[0154] [Figure 21] The in vivo efficacy of the ADCs hCl1a-LC-G2-PNU (A), hCl1f-LC-G2-PNU (B) and hCl1a(LALA)-LC-G2-PNU (C) is compared to another ADC, IMAB362-LC-G2-PNU, in the gastric cancer patient-derived tumor xenograft model GXA 3037. Each ADC was tested at doses of 0.2 mg / kg / day, 0.6 mg / kg / day or 2 mg / kg / day. Figure legend: All ADCs have PNU attached to the light chain only via the [GGGGS]-[LPQTGG]-[ethylenediamine] linker.

[0155] [Figure 22] The in vivo efficacy of hCl1a-LC-G2-PNU as an ADC is compared to the isotype control ADC Ac10-LC-G2-PNU in the colon cancer patient-derived tumor xenograft model CXF 742. Each ADC was tested at a dose of 2 mg / kg / day. Figure legend: Both ADCs have PNU attached to the light chain only via the [GGGGS]-[LPQTGG]-[ethylenediamine] linker.

[0156] [Figure 23]The in vivo efficacy of the ADCs hCl1a-LC-G2-PNU (A) and hCl1a(LALA)-LC-G2-PNU (B) is compared to another ADC, IMAB362-LC-G2-PNU, in the pancreatic cancer patient-derived tumor xenograft model PAXF 2175. Each ADC was tested at a dose of 0.2 mg / kg / day or 0.6 mg / kg / day. Figure legend: Both ADCs have PNU attached to the light chain only via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker.

[0157] [Figure 24] The in vivo efficacy of hCl1a-LC-G2-PNU as an ADC is compared to Ac10-LC-G2-PNU, an isotype control ADC, in the lung cancer patient-derived tumor xenograft model LIXFC 2050. Each ADC was tested at a dose of 2 mg / kg / day. Figure legend: Both ADCs have PNU attached to the light chain only via a [GGGGS]-[LPQTGG]-[ethylenediamine] linker.

[0158] [Diagram 25] ADCs are shown diagrammatically in which PNU is linked only to the light chain of an antibody via a spacer element -GGGGS-, an oligopeptide linker element -LPQTGG-, and a non-cleavable linker element EDA attached to C13 of PNU. Legend: In both ADCs, PNU is linked only to the light chain via the [GGGGS]-[LPQTGG]-[ethylenediamine] linker. EXAMPLES

[0159] Example 1: Generation of chimeric and humanized antibodies The techniques for generating monoclonal antibodies are already well established. The Handbook of Therapeutic Antibodies, Second Edition (2014) provides ample information on such techniques, including the generation of monoclonal antibodies by immunization of mice or rats (Moldenhauer, 2014), humanization of monoclonal antibodies (Saldanha, 2014), bioinformatics tools for analyzing antibodies (Martin and Allemn, 2014), and the development and production of therapeutic antibodies (Jacobi et al., 2014). Briefly, monoclonal antibodies against CLDN18.2 were generated by DNA immunization of rats with a plasmid encoding human CLDN18.2 cDNA (huCLDN18.2) (NCBI reference sequence: NM_001002026.3). The specific reactivity of rat immune sera against huCLDN18.2 was analyzed by flow cytometry (FC analysis) and ELISA. Next, hybridoma clones were generated from lymphocytes isolated from the immunized rats to obtain chimeric antibodies. Three clones specific to CLDN18.2 were identified, and chimeric antibodies with similar CDRs were obtained from these clones, which were named cCl1-1, cCl1-2, and cCl1-3 (see Table 3). Next, the cCl1-1, cCl1-2, and cCl1-3 antibodies were humanized, resulting in 10 humanized clones, which were named hCl1a, hCl1b, hCl1c, hCl1d, hCl1e, hCl1f, hCl1g, hCl1h, hCl1i, and hCl1j, respectively (see Table 3). These antibodies were also used to generate ADCs.

[0160] As a control, the IMAB362 antibody was synthesized from the heavy chain sequence (SEQ ID NO: 55) and the light chain sequence (SEQ ID NO: 56) according to the method published in WO2013 / 174509, named monoclonal antibody 182-D1106-362, and deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B 38124 Braunschweig, Germany) under the accession number DSM ACC2810 on October 26, 2006. [Table 3] TIFF2024500242000022.tif254168TIFF2024500242000023.tif239169TIFF2024500242000024.tif254165TIFF202 4500242000025.tif254164TIFF2024500242000026.tif240169TIFF2024500242000027.tif254165TIFF2024500242 000028.tif214169TIFF2024500242000029.tif254165TIFF2024500242000030.tif254169TIFF2024500242000031. tif245169TIFF2024500242000032.tif254166TIFF2024500242000033.tif245169TIFF2024500242000034.tif89169

[0161] The antibodies described in Examples 2 to 5 below were engineered to contain an LPQTGG tag (SEQ ID NO: 131) at the C-terminus of the heavy chain and / or a GGGGSLPQTGG tag (SEQ ID NO: 132) at the C-terminus of the light chain. In these antibodies, the C-terminal lysine (K) of the heavy chain was replaced with an arginine (R) from this tag. The addition of these tags did not alter the affinity and specificity of each antibody for CLDN18.2.

[0162] Example 2: ELISA assay and flow cytometry titration to confirm binding of chimeric and humanized antibody variants to CLDN18.2 Using CLDN18.2-containing lipoparticles as a source of antigen, the binding affinity of chimeric and humanized antibodies (hCl) against CLDN18.2 was tested in an ELISA assay. CLDN18.2 lipoparticles or null lipoparticles (as a negative control with no antigen bound to the surface) were coated onto 96-well plates to a final concentration of 10 U / ml. Plates were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with PBS containing 3% BSA for at least 1 h at 37°C. Each test antibody was serially diluted 1:3 from a starting concentration of 2 μg / ml and added to each coated well and incubated at 37°C for at least 1 h. HRP-conjugated goat anti-human secondary antibody was then bound, followed by SIGMAFAST as a peroxidase substrate. TM Plate-bound antibodies were detected by developing with OPD, stopping the reaction with 2M H2SO4, and reading the absorbance at 490 nm using an ELISA plate reader. Representative binding curves are shown in Figure 1. All of the antibodies of the present invention tested specifically bind to CLDN18.2-containing lipoparticles. Interestingly, humanization of the chimeric antibodies did not result in a loss of affinity as expected, and six of the ten antibodies showed enhanced affinity compared to the parent chimeric cCl1-1 antibody.

[0163] CLDN18.2-overexpressing PA-TU-8988S cells (Creative Bioarray, Catalog No. CSC-C0326) and CLDN18.2-overexpressing HEK293T cells (ATCC, CRL-3216 TM) was used to test the binding of chimeric and humanized antibodies to CLDN18.2 by flow cytometric titration. Flow cytometric titration allows the measurement of the half maximal effective concentration (EC50) of the tested antibody. PA-TU-8988S cells with high CLDN18.2 expression were selected by FACS. Herein, the selected cells are referred to as PA-TU-8988S-High cells. Specifically, staining of the PA-TU-8988S cell population with the IMAB362 antibody and analysis by FACS showed high and moderate expression of CLDN18.2 (see Figure 2A). To obtain a more homogenous cell population, PA-TU-8988S cells were sorted by FACS to select only cells with high CLDN18.2 expression. Briefly, PA-TU-8988S cells were suspended in FACS buffer (PBS containing 2% FCS) and incubated with 2 μg / ml IMAB362 on ice for 30 min. After washing with FACS buffer, PE-labeled Fcγ-specific goat IgG anti-human secondary antibody (eBioscience) was added and the cells were incubated on ice for 30 min. After washing, stained cells were resuspended in FACS buffer and analyzed by FACSAria. TM Cells expressing moderate levels of CLDN18.2 were removed from CLDN18.2-high cells by analyzing and sorting with the instrument (Figure 2B). After sorting, the collected PA-TU-8988S-High cells were resuspended in growth medium, grown in growth medium, and aliquots were cryopreserved in liquid nitrogen. We also generated HEK293T cells overexpressing CLDN18.2 or CLDN18.1 as described in Example 3, and analyzed the expression of CLDN18.2 by flow cytometry (Figure 3).

[0164] To quantify the binding of each antibody to CLDN18.2, 250 × 10 HEK293T cells or PA-TU-8988-High cells overexpressing CLDN18.2 were plated in FC buffer (PBS containing 2% FBS) in a 96-well plate. 3Cells were seeded at a density of 1000 cells / well and pelleted by centrifugation. IMAB362 antibody and each hCl antibody to be tested were diluted to 20 μg / ml, then serially diluted 1:4, added to the plated cells, and incubated at 4°C for 30 minutes. After washing with FC buffer, a PE-labeled anti-human IgG antibody was added as a secondary antibody to the cells, incubated at 4°C for another 30 minutes, and washed again with FC buffer. Cells were resuspended in 100 μl of FC buffer and analyzed by FACSCalibur. TM The measurements were performed using a cell analyzer (BD Biosciences, USA). The flow cytometry analysis (see Figure 5 and Table 4) showed that all hCl antibodies had higher EC50 values ​​than IMAB362, but had maximum MFI values ​​in a similar range to IMAB362. The similar maximum MFI values ​​suggest that the binding / dissociation rates of IMAB362 and hCl antibodies are also similar. [Table 4]

[0165] Example 3: Generation of pre-B L11, BxPC-3 and HEK293T cells stably expressing hCLDN18.1 or hCLDN18.2; testing of binding specificity of chimeric and humanized antibodies Pre-B cell L11 cell line (Waldmeier et al., 2016), BxPC-3 (ATCC CRL-1687 TM ) cell line, HEK293T (ATCC CRL-3216 TM ) cell lines and A549 (ATCC CCL-185 TM) cell lines do not endogenously express CLDN18.1 or CLDN18.2. Therefore, we genetically overexpressed CLDN18.1 or CLDN18.2 in HEK293T and A549 cell lines and tested antibody binding. Specifically, a transposase expression construct (pcDNA3.1-hy-mPB), a transposon construct containing the full-length huCLDN18.1 (pPB-Puro-huCLDN18.1) or a transposon construct containing the full-length huCLDN18.2 (pPB-Puro-huCLDN18.2), a puromycin resistance cassette, and an EGFP-containing construct (pEGFP-N3) as a transfection control were co-transfected into pre-B cell L11 or HEK293T cell lines by electroporation (Waldmeier et al., 2016). After electroporation, L11 cells were allowed to recover for 2 days at 37°C in growth medium in a humidified incubator under a 7.5% CO2 atmosphere, and HEK293T and A549 cells under a 5% CO2 atmosphere. Transfection was confirmed by analyzing the expression of EGFP by flow cytometry. Cells expressing CLDN18.1 or CLDN18.2 were then selected by adding 1 μg / ml puromycin to the cultures, and the selected cells were further expanded to generate frozen stocks in FCS containing 10% DMSO. Expression of CLDN18.1 or CLDN18.2 in the transfected cells was analyzed by flow cytometry (see Figure 3). Briefly, trypsinized suspension-cultured HEK293T, A549 or L11 cells were harvested by centrifugation, resuspended in PBS containing 2% FCS and stained for CLDN18.2 for 30 min on ice using 2 μg / ml IMAB362 as the primary antibody. After washing with PBS containing 2% FCS, cells were stained for 30 min on ice using PE-labeled anti-human IgG (Fcγ-specific) goat antibody (eBioscience) as the secondary antibody. Stained cells were washed again, resuspended in ice-cold Fc buffer and analyzed by FACSCalibur. TMThe cells were analyzed by the instrument (see Figures 4 and 5). Parental cells, which were not transfected and therefore do not express CLDN18.2, were used as a negative control. Analysis of CLDN18.1 expression was performed in a similar manner using a commercially available pan-CLDN18 antibody that recognizes CLDN18.1 and CLDN18.2 (see Figure 3). Any pan-CLDN18 antibody for flow cytometry measurement can be suitably used, such as anti-claudin 18 / CLDN18 (C-terminus) antibody (catalog No. AP50944PU-N) commercially available from OriGene Technologies, CLDN18 (C-terminus) rabbit polyclonal antibody (catalog No. MBS8555451) commercially available from MyBioSource, or CLDN18 antibody (catalog No. 63-847) commercially available from ProSci.

[0166] L11 or HEK293T cells stably expressing huCLDN18.1 or huCLDN18.2 were finally selected, and these cells were used to test the chimeric antibodies cCl1-1, cCl1-2, and cCl1-3 and each humanized antibody for binding specificity of binding to CLDN18.2 but not to CLDN18.1. The cells were stained with 2 μg / ml of each antibody for 30 min on ice, washed with PBS containing 2% FCS, and then stained with PE-labeled anti-human IgG (Fcγ-specific) goat antibody (eBioscience) as the secondary antibody for 30 min on ice. All three chimeric antibodies (Figure 4) and each humanized antibody (Figure 5) bound to huCLDN18.2 expressed in L11 or HEK293T cells, but not to huCLDN18.1. Furthermore, the binding of each humanized antibody to huCLDN18.2 showed an affinity comparable to that of IMAB362 and at least as good as that of cCl1-1 (FIG. 5).

[0167] Example 4: Flow cytometry test of the binding activity of humanized CLDN18.2 antibody to live tumor tissue and live gastric tissue A549 (ATCC CCL-185 TM) cell lines do not endogenously express CLDN18.1 or CLDN18.2. Therefore, we expressed CLDN18.2 in A549 cells and tested the binding of antibodies to CLDN18.2. Specifically, a transposase expression construct (pcDNA3.1-hy-mPB) (Klose et al., 2017), a transposon construct containing the full-length huCldn18.2 (pPB-Puro-huCldn18.1), a puromycin expression cassette, and an EGFP-containing construct (pEGFP-N3) as a transfection control were co-transfected into A549 cells by electroporation (Waldmeier et al., 2016). After electroporation, cells were allowed to recover for 2 days in growth medium at 37 °C in a humidified incubator under a 5% CO2 atmosphere. Transfection was confirmed by analyzing EGFP expression by flow cytometry. Cells expressing CLDN18.1 or CLDN18.2 were then selected by adding 1 μg / ml puromycin to the culture, and the selected cells were further expanded to generate frozen stocks in FCS containing 10% DMSO. Expression of CLDN18.2 in transfected A549 cells was analyzed by flow cytometry. Briefly, trypsinized A549 cells were harvested by centrifugation, then resuspended in PBS containing 2% FCS, and stained for CLDN18.2 with 2 μg / ml IMAB362 as the primary antibody for 30 min on ice. After washing the cells with PBS containing 2% FCS, they were stained with 2.5 μg / ml PE-labeled anti-human IgG (Fcγ-specific) goat antibody (eBioscience) as the secondary antibody for 30 min on ice. The stained cells were washed again, then resuspended in ice-cold FCS buffer and analyzed by FACSCalibur. TMThe CLDN18.2-expressing A549 cell line was analyzed using the ELISA kit (see FIG. 6). Parental cells that were not transfected and therefore did not express CLDN18.2 were used as a negative control. The CLDN18.2-expressing A549 cell line was deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B 38124 Braunschweig, Germany) on December 6, 2019 and is available under the accession number DSM ACC3360.

[0168] 1 × 10 CLDN18.2-expressing A549 cells suspended in 100 μl of 50% Matrigel 6 The individual pieces were subcutaneously implanted into two Balb / c mice, and the tumors were allowed to grow to 150–450 mm 3Tumor growth was observed for several weeks until it reached the desired size of . Normal gastric and tumor tissues were harvested for flow cytometry analysis. Harvested tissues were minced and digested with Miltenyi tumor dissociation kit (MACS Miltenyi Biotec, Germany). Tissue fragments were placed in dissociation buffer (prepared according to the manufacturer's instructions) in a 6-well plate and incubated at 37°C for 30 min with constant gentle shaking. Samples were resuspended, filtered through a 70 μm cell strainer (Corning, USA) and washed with 20 ml of FC buffer (PBS + 2% FBS). The resulting cell suspension was centrifuged (4°C, 400 × g for 5 min) and the supernatant was removed. If necessary, after repeated filtration and centrifugation of the cell suspension through the cell strainer, the pellet was resuspended in 5 ml of hemolysis buffer (Biolegend, USA) and incubated on ice for 4 min. After incubation, 25 ml of PBS was added and the suspension was centrifuged again (400 × g for 5 min at 4 °C). The pellet was resuspended in FC buffer (0.5–3 ml depending on the size of the pellet). The cells were transferred to a 96-well plate so that the number of cells in each well was equal, and further processed for flow cytometry analysis. Specifically, the transferred cells were washed with PBS and centrifuged (4 °C, 400 × g for 2 min). The pellet was resuspended in each well with 50 μl per well of a staining mix consisting of each selected antibody (cCl1-1, hCl1a, hCl1b, hCl1c or hCl1f at 4 μg / ml; IMAB364 at 2 μg / ml) and AF488-labeled pan-cytokeratin (AE1 / AE3) antibody (Thermo Fisher Scientific, USA) diluted in PBS, and incubated on ice for 25 min. After incubation, cells were washed twice with PBS and centrifuged (400×g for 2 min at 4°C). The pellet was resuspended in each well with 50 μl of secondary staining mix (PBS+PE-labeled anti-human antibody) (Thermo Fisher Scientific, USA) per well and incubated on ice for 25 min. After incubation, cells were washed twice again with PBS. The pellet was resuspended in 100 μl of PBS containing DAPI.Plates were stored on ice until flow cytometry analysis was performed. For flow cytometry analysis, live cells were separated from dead cells by forward scatter and DAPI staining. Live cells were then gated for the presence of cytokeratin (AF888 positive) and binding of CLDN18.2 antibody (PE positive cells). The results of flow cytometry analysis are shown in Figure 7 and Table 5. These results are the average of data from two mice.

[0169] All of the tested antibodies (cCl1-1, hCl1a, hCl1b, hCl1c, hCl1f and IMAB364) bound to CLDN18.2-expressing tumor cells at similar rates of about 20% to 30%. Surprisingly, only IMAB362 showed binding to normal gastric cells expressing CLDN18.2, and cCl1-1, hCl1a, hCl1b, hCl1c or hCl1f showed barely detectable binding to normal gastric cells at less than 1%. The difference in the binding ability of the antibodies between CLDN18.2 expressed in tumor cells grown from injected CLDN18.2-expressing A549 cells and CLDN18.2 expressed in normal gastric cells was also expressed as the ratio of the percentage of positive tumor cells divided by the percentage of positive gastric cells (see the rightmost column in Table 5). This ratio was less than 5, averaging around 1, for IMAB362, but greater than 15, averaging greater than 30, for the humanized cCl1-1 clones tested (hCl1a, hCl1b, hCl1c, and hCl1f). [Table 5]

[0170] Thus, the tested humanized clones of cCl1-1 (hCl1a, hCl1b, hCl1c, and hCl1f) and cCl1-1 bound more strongly to tumor cells than to normal gastric cells, indicating that these antibodies are tumor-specific CLDN18.2 antibodies, whereas IMAB362 could not distinguish between CLDN18.2-expressing tumor cells and CLDN18.2-expressing normal gastric cells.

[0171] Example 5: Testing of humanized CLDN18.2 antibodies by immunohistochemistry (IHC) of frozen tissue samples 1 × 10 A549 cells expressing CLDN18.2 6 Fresh gastric and tumor tissue samples expressing CLDN18.2 were collected from Balb / c mice subcutaneously implanted with 1000 ng / mL IgG, placed in appropriate tissue embedding dishes, and snap frozen in OCT compound. The tissue was thin-sectioned into 5-15 μm thick sections at -20°C using a cryostat, mounted on microscope slides at room temperature, and frozen until IHC staining. Before staining, the slides were brought to room temperature and fixed in pre-cooled acetone (-20°C) for 10 min. After acetone evaporation at room temperature, the slides were rinsed with TBS to block nonspecific staining sites. Specifically, the slides were incubated in 0.3% H2O2 at room temperature for 15 min, washed with TBS, and incubated at room temperature for 60 min with a drop of peroxidase blocking solution (Agilent, USA). After blocking, the slides were stained with antibodies. Specifically, primary antibodies (hCl1a, hCl1b, hCl1c, hCl1f, IMAB362 or pan-CLDN18 antibody [34H14L15] (Abcam, USA) were applied to the slides and incubated at room temperature for 120 min, then washed with TBS, and HRP-conjugated anti-human antibody (or anti-rabbit antibody for pan-CLDN18 antibody) was applied to the slides and incubated at room temperature for 30 min. Binding to tissue sections was performed by treating the slides with DAB+substrate chromogen system (Agilent, USA) according to the manufacturer's instructions. The combined CLDN18.2 or pan-CLDN18 antibodies were detected. After washing with TBS, the slides were counterstained with hematoxylin, rinsed with dH2O for 15 min, dehydrated by successive washes with 95% ethanol and 100% ethanol, and cleared with xylene. The slides were then coverslipped using glycerol mounting medium (Agilent, USA). Representative micrographs of staining of mouse normal stomach tissue and mouse tumor tissue are shown in Figure 8 and Figure 9, respectively.

[0172] Figure 8 shows representative staining of normal gastric tissue. In tissues co-stained with hCl1a, hCl1b, hCl1c or hCl1f (shown in panels A, B, C and D, respectively), only nuclear hematoxylin staining can be confirmed. On the other hand, in tissues co-stained with IMAB362 (panel E), CLDN18.2 expressed in membranous structures is stained with DAB. Thus, unlike IMAB362, which binds to normal gastric tissues expressing CLDN18.2, the humanized clones of cCl1-1 tested (hCl1a, hCl1b, hCl1c and hCl1f) were shown not to bind to normal gastric tissues expressing CLDN18.2. Furthermore, Figure 9 shows representative staining of tumor tissue. Representative photographs of tumor tissues stained with hCl1a, hCl1f, IMAB362 or Abcam's pan-CLDN18 antibody [34H14L15] are shown in panels A, B, C and D, respectively. In tumors stained with the tested antibodies, CLDN18.2 expressed in membranous structures was strongly stained with DAB. The tested humanized clones of cCl1-1 (hCl1a and hCl1f) bound to mouse tumor tissues expressing CLDN18.2 to the same extent as IMAB362 and pan-CLDN18 antibodies. Thus, the humanized clones of cCl1-1 bind more strongly to tumor tissues expressing CLDN18.2 than to normal gastric tissues expressing CLDN18.2.

[0173] Example 6: Analysis of asparagine deamidation and aspartic acid isomerization tendencies of humanized antibody (hCl) variants and IMAB362 During biopharmaceutical manufacturing, storage or clinical application (in vivo), deamidation of asparagine (N) residues and isomerization of aspartic acid (D) residues can occur. Deamidation and isomerization can alter protein structure, function, activity, stability and immunogenicity. Therefore, such changes need to be minimized and controlled, especially with regard to drug regulations. The presence of asparagine deamidation and aspartic acid isomerization motifs can be analyzed in silico. The most common asparagine deamidation motif is the NG motif, and the most common aspartic acid isomerization motif is the DG motif.

[0174] In silico analysis revealed that both hCl antibodies have a DG motif in the second CDR of the VL region that may lead to aspartic acid isomerization, but neither hCl antibody nor IMAB362 have an NG motif in the CDR that may lead to deamidation. To confirm the predictions from in silico analysis, hCl antibody and IMAB362 were stressed with high or low pH and heat to accelerate amino acid changes that may occur during manufacturing and long-term storage. Briefly, the buffer in the antibody samples was exchanged into 20 mM sodium phosphate buffer (pH 8.0) for the asparagine deamidation stress test or 20 mM citrate buffer (pH 5.5) for the aspartic acid isomerization stress test using Amicon centrifugal filters, and each sample was diluted to a final concentration of 3.0 mg / ml. Thirty microliters of samples were incubated at 40°C for 1 week (asparagine deamidation) or 2 weeks (aspartic acid isomerization) in a block bath with a heated lid to prevent concentration. Stressed and unstressed samples were stored at -80°C. Samples were analyzed for asparagine deamidation and aspartic acid isomerization by strong cation exchange (SCX) chromatography. Asparagine deamidation is indicated by an increase in the peak area (bM) before the main peak in the SCX chromatogram, whereas aspartic acid isomerization is indicated by an increase in the peak area (aM) after the main peak in the SCX chromatogram (Du et al., 2012). SCX chromatography was performed on a MAbPac SCX-10 column (Thermo Fisher Scientific, Basel, Switzerland) using buffer A (pH 4.0) and buffer B (pH 11.0). The flow rate was 0.5 ml / min with a pH gradient of 30-80% buffer B. 10 μg of sample was added to 20 μl of buffer A and injected into the column. Sample detection was performed by measuring the protein absorbance at 280 nm. The bM value of each hCl antibody increased only by about 27.9-32.2% (see Table 6), which was not considered to be of particular importance.On the other hand, in the case of IMAB362, the bM value increased significantly to 40.9% (see Table 6) despite the absence of an NG motif in the variable domain. Unlike the anti-CLDN18.2 monoclonal antibody of the present invention, IMAB362 has two NS motifs, located in CDR3 (amino acids 103-104) of the heavy chain (SEQ ID NO: 55) and CDR1 (amino acids 31-32) of the light chain (SEQ ID NO: 56). The NS motif is the motif with the second highest tendency for deamidation. [Table 6]

[0175] The effect of the asparagine deamidation stress test on the binding affinity of the hCl1a, hCl1i or IMAB362 antibodies to CLDN18.2 was tested by an ELISA assay using CLDN18.2-containing lipoparticles as the antigen source. CLDN18.2 lipoparticles or null lipoparticles (without antigen) were coated onto 96-well plates to a final concentration of 10 U / ml in 100 mM sodium carbonate (pH 9.6). Plates were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with PBS containing 3% BSA for at least 1 h at 37°C. Each hCl antibody was serially diluted 1:3 from a starting concentration of 2 μg / ml and incubated at 37°C for at least 1 h. Plate-bound antibodies were then detected by binding HRP-conjugated goat anti-human secondary antibody, developing with Sigma-Fast OPD as peroxidase substrate, stopping the reaction with 2M H2SO4, and reading absorbance at 490 nm using an ELISA plate reader. The EC50 value of IMAB362 was 1.8-fold higher after the deamidation stress test (unstressed control: EC50=51.5ng / ml, stressed: EC50=95.09ng / ml) (see FIG. 10). This result may be related to the increase in bM to 40.9% in SCX chromatography after the deamidation stress test (see Table 6). Supporting the results of the asparagine deamidation stress test by SCX chromatography, no significant difference was observed in the binding of hCl1a and hCl1i antibodies to the antigen after the deamidation stress test (see Table 6). Thus, the deamidation stress test showed that the hCl antibody not only has a lower tendency to deamidate than IMAB362, but also has a lower tendency to lose binding to the target compared to IMAB362, and is more stable than IMAB362 during manufacturing, storage, and clinical application (in vivo), as expected. This indicates that the use of hCl antibody can provide a more homogeneous and active antibody / product than IMAB362.

[0176] All of the hCl antibodies have DG motifs in the second CDR of the VL region and the CH2 and CH3 domains of the heavy chain (VL-CDR2 (62nd position), CH2 (282nd position), CH3 (403rd position)) that may cause aspartic acid isomerization. However, contrary to the results expected from the report by Du et al. (Du et al., 2012), no aspartic acid isomerization was observed even when the aspartic acid isomerization stress test was performed (see Table 7). The aM values ​​of the non-stressed samples were significantly higher from the beginning (except for IMAB362). This may be due to the fact that the heavy chain lysine is a truncated variant. Only the IMAB362 antibody did not show a high aM value in the non-stressed samples. Among the antibodies tested, the IMAB362 antibody was the only anti-CLDN18.2 antibody that did not have a lysine at the C-terminus, therefore, the truncation of the C-terminal lysine of the hCl antibody was most likely the reason for the high aM values ​​in both non-stressed and stressed samples. [Table 7]

[0177] Example 7: Conjugation of glycine-modified toxic drugs to monoclonal antibodies using sortase-mediated conjugation to form ADCs Sortase A enzyme: Recombinant sortase A enzyme from Staphylococcus aureus was produced in E. coli and affinity purified in a manner similar to that disclosed in WO2014140317A1.

[0178] Generation of glycine-modified toxic drugs: The diglycine-modified EDA-anthracycline derivative, GG-EDA-PNU-159682 (see also FIG. 25), was produced by Levena Biopharma, San Diego, USA. The toxic drug, PNU-159682, was synthesized to contain the non-cleavable linker EDA and the oligopeptide linker GG. The identity and purity of the glycine-modified toxic drug were confirmed by mass spectrometry and HPLC. The purity of the glycine-modified toxic drug was determined by HPLC chromatography and was greater than 95%.

[0179] Sortase-based antibody conjugation: The above toxic drugs were bound to the LPQTG-tagged anti-CLDN18.2 antibody consisting of a heavy chain and a light chain or only a light chain shown in Table 3, or to a comparative antibody (IMAB362 and CD30-specific antibody AC10). In addition, only the light chain of the antibody shown in Table 3 was bound to this toxic drug. The binding of the antibody and the toxic drug was carried out as follows. First, 20 μM of the LPQTG-tagged monoclonal antibody consisting of a heavy chain and a light chain or only a light chain, 100 μM of the glycine-modified toxic drug, and 4 μM of sortase A were added to a binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM CaCl2, 10% glycerol), and incubated at 25° C. for 3.5 hours or at 4° C. overnight. The reaction was stopped by passing the reaction solution through a rProtein A GraviTrap column (GE Healthcare). The column was washed with 36 column volumes of wash buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10% (v / v) glycerol). The bound complex was eluted with 5 column volumes of elution buffer (0.1 M glycine pH 2.7, 50 mM NaCl, 10% (v / v) glycerol) and the eluate was aliquoted in 0.5 column volumes into tubes containing 1 M HEPES (pH 8) to neutralize the acid. Protein-containing fractions were pooled and run on a Zeba spin desalting column (Thermo Fisher) with histidine buffer (15 mM histidine, pH 6.5, 175 mM sucrose, 0.02% Tween 20). Endotoxin was further removed with Pierce High Capacity Endotoxin Removal Resin (Thermo Fisher) and sterile filtered through a 0.22 μm filter. The final concentration of each ADC was determined by UV-Vis spectroscopy.

[0180] In addition, as another ADC, IMAB362-MC-vc-PAB-MMAE was prepared in a manner similar to that disclosed in WO2016 / 166122 (Example 1, Section 3, pages 75-76).

[0181] ADC Analysis: Drug-to-antibody ratio (DAR) was assessed by reversed-phase chromatography using a PLRP-S column (Agilent) with pore size 300 Å, size 2.1 × 150 mm, and particle size 3 μm. Analysis was performed at 60 °C and 0.7 ml / min with a 9 min linear gradient (25-40%) between 0.1% TFA / 3% CH3CN / H2O and 0.1% TFA / CH3CN, followed by a 4 min linear gradient (40-75%) hold. Samples were first reduced by incubation in 10% v / v 0.5 M DTT (pH 8.0) for 15 min at 37 °C. All generated ADCs had a DAR LC=2 or DAR HC-LC=4.

[0182] Example 8: In vitro cytotoxicity assay of ADCs using anti-CLDN18.2 antibodies in CLDN18.2-expressing cells (data taken from NBET'2483) In Example 8 and Example 9 described later, the ADC represented by [antibody]-HC-LC-PNU is an ADC in which the toxic drug PNU-159682 is bound to the heavy and light chains of an antibody, and has a DAR of 4. The ADC represented by [antibody]-HC-PNU or [antibody]-LC-PNU is an ADC in which the toxic drug PNU-159682 is bound to the heavy or light chain of an antibody, and has a DAR of 2. All of these ADCs contain the oligopeptide linker -LPQTGG- and the non-cleavable linker ethylenediamine. The structure of the ADC represented by [antibody]-LC-PNU is shown in FIG. 25.

[0183] The cytotoxicity of each ADC with anti-CLDN18.2 was examined using A549 cells, HEK293T cells or BxPC-3 cells genetically engineered to overexpress hCLDN18.2 (see Examples 3 and 4), or PA-TU-8988S-high cells that endogenously express hCLDN18.2 (see Example 2), and compared with IMAB362-HC-G2-PNU, IMAB362-LC-G2-PNU, IMAB362-HC-LC-G2-PNU or IMAB362-MC-vc-PAB-MMAE. To confirm specificity for CLDN18.2 but not CLDN18.1, HEK293T cells and A549 cells genetically engineered to overexpress hCLDN18.1 (see Example 3) were also used.

[0184] Briefly, 1000 A549 or HEK293T cells / well, 5000 BxPC-3 cells / well, or 10000 PA-TU-8988S-high cells / well were seeded into 96-well white clear-bottom plates (Greiner) in 75 μl of high glucose DMEM, 10% FCS, 100 IU / ml penicillin / streptomycin / fungizone, and 2 mM L-glutamine (except for the water-filled wells at the edge of the plate) and cultured at 37°C in a humidified incubator with a 7.5% CO2 atmosphere. After 1 day of culture, 25 μl of each ADC was added to the wells in a 4-fold serial dilution in complete growth medium. The concentration of each ADC was 5000-0.076ng / ml for A549 cells, 1000-0.015ng / ml for HEK293-T cells, 20000-0.25ng / ml for BxPC-3 cells, and 20000-0.31ng / ml for PA-TU-8988S cells. After another 4 days, the plate was removed from the incubator and returned to room temperature. After about 30 minutes, 50μl of CellTiter-Glo (registered trademark) 2.0 luminescence reagent (Promega) was added to each well. The plate was shaken at 450 rpm for 5 minutes, then incubated for 10 minutes without shaking, and luminescence measurements were performed using a Tecan Spark 10M plate reader with an integration time of 250ms per well. The amount of luminescence and the ADC concentration (ng / ml) were plotted, and curve fitting was performed using Graphpad Prism software (see Figures 11 to 19).

[0185] As a result of in vitro cytotoxicity assays, in CLDN18.2-overexpressing HEK293T cells (see Figure 11), CLDN18.2-overexpressing BxPC-3 cells (see Figure 13), CLDN18.2-overexpressing A549 cells (see Figure 14), and PA-TU-8988S-High cells (see Figure 16), ADCs in which PNU was conjugated only to the heavy chain of cCl1-1, cCl1-2, or cCl1-3, ADCs in which PNU was conjugated only to the light chain of these antibodies, or ADCs in which PNU was conjugated to both the heavy and light chains of these antibodies showed superior cytotoxic activity to ADCs in which IMAB362 and PNU were conjugated in the same manner and IMAB362-MC-vc-PAB-MMAE. On the other hand, cytotoxic activity was observed only at very high concentrations of ADC in CLDN18.1-overexpressing HEK293T cells (see FIG. 12) and CLDN18.1-overexpressing A549 cells (see FIG. 15). Cytotoxic activity against CLDN18.1-overexpressing cells was due to at least 1000-fold higher concentrations of toxic drug and was observed only when conjugated at DAR=4 (when toxic drug is conjugated to both heavy and light chains of antibody). Similarly, control ADC using Ac10 antibody, which does not target CLDN18.2, also showed cytotoxic activity only at very high concentrations of ADC (see FIG. 14 and FIG. 15).

[0186] In addition, in vitro cytotoxicity assays showed that ADCs in which toxic drugs were bound only to the light chains of the hCl1a to hCl1j antibodies (ADCs with DAR = 2) showed superior cytotoxic activity to ADCs in which toxic drugs were bound only to the light chains of IMAB362 in A549 cells overexpressing CLDN18.2 (see Figure 17), HEK293T cells overexpressing CLDN18.2 (see Figure 18), and PA-TU-8988S cells (see Figure 20). ADCs using the hCl1a to hCl1j antibodies selectively exhibited cytotoxic activity against cells overexpressing CLDN18.2, but did not exhibit cytotoxic activity against HEK293T cells overexpressing CLDN18.1 (see Figure 19).

[0187] EC of humanized antibody with PNU bound only to the light chain 50 The values ​​are shown in Table 9. EC 50 The value is the EC 50 The analytical function "log(inhibitor) vs. response - variable slope (4 parameters)" was used. [Table 9]

[0188] Overall, all of the ADCs of the present invention exhibited higher cytotoxic activity than IMAB362-LC-G2-PNU, demonstrating that they exert high cytotoxicity in vitro.

[0189] Example 9: Analysis of the in vivo efficacy of hCl1a-LC-G2-PNU and hCl1f-LC-G2-PNU as ADCs in patient-derived tumor xenograft models The following studies were performed at Charles River GmbH (Freiburg, Germany). [Table 10]

[0190] The efficacy of the anti-CLDN18.2 antibody-based ADCs hCl1a-LC-G2-PNU, hCl1a(LALA)-LC-G2, and hCl1f-LC-G2-PNU was investigated using patient-derived tumor xenograft (PDX) models according to the following study protocol. [Table 11]

[0191] Mice were implanted subcutaneously on one side with PDX material. When tumors reached the randomization criteria, mice were divided into groups and administered ADC or vehicle a total of three times as shown in Table 11. Tumor volumes were measured using calipers and body weights were recorded twice weekly. Tumor volumes were >2000 mm 3 Mice were euthanized when they reached a body weight of 100 mg / kg or showed significant weight loss (overall weight loss of >30% or >20% over a 2-day period).

[0192] Figures 20 to 23 show the relative changes in tumor volume in various PDX models during the study period. Tumor xenografts formed from tumor material derived from patients expressing CLDN18.2 showed a significant response to administration of the ADC of the present invention. The response (delay in tumor growth or tumor shrinkage) when the ADC of the present invention was administered at a low dose (0.2 mg / kg or 0.6 mg / kg) was better than when the ADC of the same binding mode using IMAB362, an anti-CLDN18.2 antibody, was administered at the same dose, and when administered at a high dose of 2 mg / kg, it was as good as the ADC of the same binding mode using IMAB362.

[0193] Various embodiments 1.General formula A-(LT) n or a pharma- ceutically acceptable salt or ester thereof, aA is an antibody or fragment thereof that binds to CLDN18.2, comprising an HCDR1 sequence of SEQ ID NO: 21, an HCDR2 sequence of SEQ ID NO: 22, and an HCDR3 sequence of SEQ ID NO: 23, and an LCDR1 sequence of SEQ ID NO: 24, an LCDR2 sequence of SEQ ID NO: 25, and an LCDR3 sequence of SEQ ID NO: 26; bL is a linker, cT is an anthracycline as a toxic drug; n is an integer from 1 to 10; Antibody-drug conjugates.

[0194] 2. The antibody-drug conjugate of embodiment 1, wherein the linker L comprises at least one non-cleavable linker element.

[0195] 3. The non-cleavable linker element is i. Ethylenediamine (EDA), jN-Formyl-N,N'-dimethylethylenediamine, k. diethylamine (DEA), l. The formula: [ka] Piperazine-derived compounds represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element, m. The formula: [ka] A compound represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element, n. The formula: [ka] A compound represented by the formula: (wherein the wavy line represents the bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof), o. The formula: [ka] A maleimidocaproyl compound represented by the formula: (wherein the wavy line indicates attachment to another linker element, and [Ab] indicates the antibody or fragment thereof), and p. The formula: [ka] A compound represented by the formula: (In the formula, the wavy line represents a bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof.) and The non-cleavable linker element is linked to the toxic drug via an amide bond or an ether bond. 3. The antibody-drug conjugate of embodiment 2, characterized in that

[0196] 4. The antibody-drug conjugate of embodiment 2 or 3, wherein the linker further comprises an oligopeptide linker element, an enzymatically cleavable linker element and / or a spacer element.

[0197] 5. One oligopeptide linker element contains the following as a sortase recognition motif: -LPXTG m -,-LPXAG m -,-LPXSG m -,-LAXTG m -,-LPXTG m -,-LPXTA m -, -NPQTG m - and - NPQTN m -, G m is an oligoglycine, where m is an integer from 1 to 21; m is oligoalanine, where m is an integer from 1 to 21; m is an oligoasparagine, where m is an integer from 1 to 21; and X is any of the conceivable amino acids; Preferably, the oligopeptide as a sortase recognition motif is -LPQTGG- or -LPETGG-. 5. The antibody-drug conjugate of embodiment 4, characterized in that

[0198] 6. The oligopeptide linker element is a. The sequence of SEQ ID NO: 131, or b. Sequence of SEQ ID NO: 132 6. The antibody-drug conjugate of embodiment 5, comprising:

[0199] 7. The enzyme-cleavable linker element has the formula: [ka] (wherein the wavy line indicates a bond to another linker element) 7. The antibody-drug conjugate of any one of embodiments 4 to 6, comprising a val-cit-PAB linker, which is a compound represented by the formula:

[0200] 8. The spacer element comprises a peptidic flexible oligopeptide, preferably the peptidic flexible oligopeptide consists of G and S, more preferably the peptidic flexible oligopeptide consists of (GGGGS) o (wherein o is 1, 2, 3, 4 or 5).

[0201] 9. a. The antibody-drug conjugate has the structure A-([oligopeptide linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [LPXTGG]-[ethylenediamine] and [ka] Selected from; b. The antibody-drug conjugate has the structure A-([oligopeptide linker element-enzyme-cleavable linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine] and ii. [LPXTGG]-[vc-PAB]-[piperazine] Selected from; c. The antibody-drug conjugate has the structure A-([spacer element-oligopeptide linker element-non-cleavable linker element]-T) n and preferably, the linker is i. [GGGGS]-[LPXTGG]-[ethylenediamine] and [ka] or d. The antibody-drug conjugate has the structure A-([spacer element-oligopeptide linker element-enzyme-cleavable linker element-non-cleavable element]-T)n and preferably, the linker is i. [GGGGS]-[LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine] and ii. [GGGGS]-[LPXTGG]-[vc-PAB]-[piperazine] Selected from: The antibody-drug conjugate according to any one of embodiments 1 to 8.

[0202] 10. The antibody drug conjugate of embodiment 9, wherein the non-cleavable linker element is ethylenediamine and the oligopeptide linker element is LPXTGG, where X is Q or E, preferably, X is Q.

[0203] 11. a. (LT) is covalently linked to both light chains of said antibody; or b. (LT) is covalently linked to both heavy chains of said antibody; or c. (LT) is covalently linked to both light chains and both heavy chains of said antibody; The antibody-drug conjugate according to any one of embodiments 1 to 10.

[0204] 12. a. (LT) is linked to the C-terminus of a light or heavy chain of the antibody; or b. (LT) is linked to an amino acid side chain of the light or heavy chain of the antibody. 12. The antibody-drug conjugate according to any one of embodiments 1 to 11,

[0205] 13. The anthracycline derivative is represented by the following formula (I): [ka] (In the formula, R1 is a hydrogen atom, a hydroxyl group, or a methoxy group, R2 is a C1-C5 alkoxy group. and wherein the non-cleavable linker element has C 13 Covalently bonded at position C 14 and the hydroxyl group is removed, or C 14 13. The antibody-drug conjugate according to any one of embodiments 1 to 12, characterized in that the antibody-drug conjugate is covalently linked to the non-cleavable linker element at a hydroxyl group at position 1 of the

[0206] 14. The antibody-drug conjugate according to any one of the preceding embodiments, characterized in that the anthracycline derivative is a derivative of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682).

[0207] 15. The antibody or fragment thereof, A, a. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 15 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; b. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; c. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16, and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:17, the LCDR2 sequence of SEQ ID NO:14, and the LCDR3 sequence of SEQ ID NO:11; d. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:18, the LCDR2 sequence of SEQ ID NO:19 and the LCDR3 sequence of SEQ ID NO:11; e. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 15 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; f. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:20 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; g. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:20, and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:18, the LCDR2 sequence of SEQ ID NO:19, and the LCDR3 sequence of SEQ ID NO:11; h. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 20 and the HCDR3 sequence of SEQ ID NO: 8, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; or i. HCDR1 sequence of SEQ ID NO: 12, HCDR2 sequence of SEQ ID NO: 20, and HCDR3 sequence of SEQ ID NO: 8, and LCDR1 sequence of SEQ ID NO: 17, LCDR2 sequence of SEQ ID NO: 14, and LCDR3 sequence of SEQ ID NO: 11 The antibody-drug conjugate according to any one of embodiments 1 to 14, comprising:

[0208] 16. The antibody or fragment thereof, A, a. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:2 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; b. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:7 and the HCDR3 sequence of SEQ ID NO:8, and the LCDR1 sequence of SEQ ID NO:9, the LCDR2 sequence of SEQ ID NO:10 and the LCDR3 sequence of SEQ ID NO:11; or c. The HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 2, and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 13, the LCDR2 sequence of SEQ ID NO: 14, and the LCDR3 sequence of SEQ ID NO: 11 The antibody-drug conjugate according to any one of embodiments 1 to 14, comprising:

[0209] 17. The antibody or fragment thereof, A, a. The VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 38; b. the VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:38; c. A VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:39; d. The VH sequence of SEQ ID NO:34 and the VL sequence of SEQ ID NO:40; e. the VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:38; f. the VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:41; g. The VH sequence of SEQ ID NO:36 and the VL sequence of SEQ ID NO:40; h. the VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:41; i. the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38; or j. The VH sequence of SEQ ID NO:37 and the VL sequence of SEQ ID NO:39 16. The antibody-drug conjugate according to any one of embodiments 1 to 15, comprising:

[0210] 18. The antibody or fragment thereof, A, a. The VH sequence of SEQ ID NO:27 and the VL sequence of SEQ ID NO:28; b. A VH sequence of SEQ ID NO:29 and a VL sequence of SEQ ID NO:30; or c. The VH sequence of SEQ ID NO:31 and the VL sequence of SEQ ID NO:32 17. The antibody-drug conjugate of any one of embodiments 1 to 14 and 16, comprising:

[0211] 19. The antibody or fragment thereof, A, a. The heavy chain sequence of SEQ ID NO:46 and the light chain sequence of SEQ ID NO:51; b. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:51; c. A heavy chain sequence of SEQ ID NO:47 and a light chain sequence of SEQ ID NO:52; d. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:53; e. a heavy chain sequence of SEQ ID NO:48 and a light chain sequence of SEQ ID NO:51; f. The heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:54; g. A heavy chain sequence of SEQ ID NO:49 and a light chain sequence of SEQ ID NO:53; h. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:54; i. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:51; or j. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:52; or These sequences in which the Fc domain was recombined 18. The antibody-drug conjugate of any one of embodiments 1 to 15 and 17, comprising:

[0212] 20. A method for producing an antibody-drug conjugate according to any one of embodiments 1 to 19, comprising: g. providing as A an antibody or fragment thereof having an oligopeptide linker element in its light and / or heavy chain, preferably at its C-terminus, and optionally a spacer element between said light and / or heavy chain and said oligopeptide linker element; h. providing as T one or more toxic drugs having a non-cleavable linker element; and i. linking the antibody and the toxic drug to obtain an antibody-drug conjugate. A method comprising:

[0213] 21. An antibody-drug conjugate comprising: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 46 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. An antibody-drug conjugate consisting of

[0214] 22. An antibody-drug conjugate comprising: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 133 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. An antibody-drug conjugate consisting of

[0215] 23. An antibody-drug conjugate comprising: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 134 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQXTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl group removed. An antibody-drug conjugate consisting of

[0216] 24. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of embodiments 1 to 23 and an additive.

[0217] 25. An antibody-drug conjugate according to any one of embodiments 1 to 23 for use in therapy.

[0218] 26. An antibody-drug conjugate according to any one of embodiments 1 to 23 for use in the treatment of cancer.

[0219] 27. The antibody-drug conjugate of embodiment 24, wherein the cancer is selected from pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer and lung cancer.

[0220] array SEQ ID NO:1 DYAMH Sequence number 2: WINTYTGKPTYADDFKG SEQ ID NO:3 AVFYGYTMDA Sequence number 4 RASEDIYSNLA SEQ ID NO:5 SVKRLQD SEQ ID NO:6 LQGSNFPLT SEQ ID NO:7 WINAYTGKPTYADDFKG SEQ ID NO:8 AVYYGYTMDA Sequence number 9: RTSEDIYSNFA SEQ ID NO:10 SVNRLQD Sequence number 11 LQGSKFPLT SEQ ID NO:12 DYAMY Sequence number 13: RTSEDIYSNLA SEQ ID NO:14 AIKRLQD SEQ ID NO:15 WINTYTGKPTYAQKFQG Sequence number 16: WINTYTGKPTYSQKFQG Sequence number 17: RTSEDIYSNLA Sequence number 18: RTSEDIYSNFA SEQ ID NO:19 SVNRLQD Sequence number 20 WINAYTGKPTYAQKFQG Sequence number 21: DYAMX The fifth X is H or Y. Sequence number 22: WINXYTGKPTYXXXFXG The fourth X is a T or an A; The 12th X is A or S; The 13th X is D or Q; The 14th X is D or K; The 16th X is K or Q. SEQ ID NO:23 AVXYGYTMDA The third X is F or Y. Sequence number 24: RXSEDIYSNXA The second X is A or T; The 10th X is L or F. SEQ ID NO:25 XXXRLQD The first X is S or A; The second X is V or I; The third X is K or N. SEQ ID NO:26 LQGSXFPLT The fifth X is K or N. SEQ ID NO:27 cCl1-1 heavy chain variable region QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMHWVKQAPGKGLKWMGWINTYTGKPTYADDFKGRFVFSLEASASTANLQISNLKNEDTATYFCARAVFYGYTMDAWGQGTSVTVSS SEQ ID NO:28 cCl1-1 Light chain variable region DIQMTQSPASLSASLGETISIACRASEDIYSNLAWYQQKSGKSPQLLIFSVKRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSNFPLTFGSGTKLEIK SEQ ID NO:29 cCl1-2 heavy chain variable region QIQLVQSGPELKKPGESVKISCKTSGYTFTDYAMHWVKQGPGKGMKWMGWINAYTGKPTYADDFKGRFVLSLEASASTANLQISNLKNEDTATYFCARAVYYGYTMDAWGQGTSVIVSS SEQ ID NO: 30 cCl1-2 light chain variable region DIQMTQSPASLSASLGETISIECRTSEDIYSNFAWFQQKSGKSPQLLIYSVNRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSKFPLTTFGSGTKLEIK SEQ ID NO:31 cCl1-3 heavy chain variable region QIQLVQSGPELKKPGESVKISCKASGYTFTDYAMYWVKQVPGKGLRWMGWINTYTGKPTYADDFKGRFVFSLEASASTANLQISNLKNEDTATYFCARAVFYGYTMDAWGQGTSVTVSS SEQ ID NO:32 cCl1-3 light chain variable region DIQMTQSPASLSASLGETISIACRTSEDIYSNLAWYQQKSGKSPQLLIFAIKRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSKFPLTFGSGTKLEIK SEQ ID NO: 33 hCL1a heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSS SEQ ID NO: 34 hCL1b, hCL1c and hCL1d heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSS SEQ ID NO: 35 hCL1e heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMYWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSS SEQ ID NO: 36 hCL1f and hCL1g heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINAYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSS SEQ ID NO: 37 hCL1h, hCL1i and hCL1j heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMYWVRQAPGQRLEWMGWINAYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVYYGYTMDAWGQGTLVTVSS SEQ ID NO: 38 hCL1a, hCL1b, hCL1e and hCL1i light chain variable region DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIFSVKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSNFPLTFGQGTKVEIK SEQ ID NO: 39 hCL1c and hCL1j light chain variable region DIQMTQSPSSLSASVGDRVTITCRTSEDIYSNLAWYQQKPGKAPKLLIFAIKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKVEIK SEQ ID NO: 40 hCL1d and hCL1g light chain variable region DIQMTQSPSSLSASVGDRVTITCRTSEDIYSNFAWYQQKPGKAPKLLIYSVNRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKVEIK SEQ ID NO: 41 hCL1f and hCL1h Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIYSVKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSNFPLTFGQGTKVEIK SEQ ID NO: 42 hCL3a, hCL3b and hCL3c heavy chain variable region QVQLQESGPGLVKPSETLSLTCAVSGYSVSSNYRWHWIRQPPGKGLEWIGYINIAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARNPSITRAMDAWGQGTLVTVSS SEQ ID NO: 43 hCL3a light chain variable region DIQMTQSPSSLSASVGDRVTITCKSSQNIFKNLEWYQQKPGKAPKLLIYYTNNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYNSGPFTFGQGTKVEIK SEQ ID NO: 44 hCL3b light chain variable region DIQMTQSPSSLSASVGDRVTITCRSSQNIFKNLEWYQQKPGKAPKLLIYYTNNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYNSGPFTFGQGTKVEIK SEQ ID NO: 45 hCL3c light chain variable region DIQMTQSPSSLSASVGDRVTITCRSSQNIFKNLEWYQQKPGKAPKLLIYYTNNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYNSGPFTFGQGTKVEIK SEQ ID NO: 46 hCL1a full length heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R SEQ ID NO: 47 hCL1b, hCL1c and hCL1d full length heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X = K or R Sequence number 48 hCL1e heavy chain full length QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMYWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X = K or R SEQ ID NO: 49 hCL1f and hCL1g full length heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINAYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R SEQ ID NO: 50 hCL1h, hCL1i and hCL1j full length heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMYWVRQAPGQRLEWMGWINAYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVYYGYTMDAWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R SEQ ID NO: 51 hCL1a, hCL1b, hCL1e and hCL1i full length light chain DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIFSVKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSNFPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:52 hCL1c and hCL1j full length light chain DIQMTQSPSSLSASVGDRVTITCRTSEDIYSNLAWYQQKPGKAPKLLIFAIKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:53 hCL1d and hCL1g full length light chain DIQMTQSPSSLSASVGDRVTITCRTSEDIYSNFAWYQQKPGKAPKLLIYSVNRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:54 hCL1f and hCL1h full length light chain DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIYSVKRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGSNFPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 55 IMAB362 full length heavy chain QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R SEQ ID NO: 56 IMAB362 full length light chain DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:57 DQWSTQDLYN SEQ ID NO:58 NNPVTAVFNYQ SEQ ID NO:59 STQDLYNNPVTAVF SEQ ID NO:60 TNFWMSTANMYTG SEQ ID NO:61 ALMIVGIVLGAIGLLV SEQ ID NO:62 RIGSMEDSAKANMTLTSGIMFIVS SEQ ID NO:63 METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLGTELGSTPVWWNSADGRMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRAAIQHSGGRSRRARTKTHLRRGSE SEQ ID NO:64 MDQWSTQDLYNNPVT SEQ ID NO:65 LYNNPVTAVFNYQGL SEQ ID NO:66 VFNYQGLWRSCVRES SEQ ID NO:67 QGLWRSCVRESSGFT SEQ ID NO:68 RSCVRESSGFTECRG Sequence number 69: TEDEVQSYPSKHDYV Sequence number 70 EVQSYPSKHDYV SEQ ID NO:71 gactacgcgatgcac SEQ ID NO: 72 tggatcaacacgtacacggggaagccgacatacgcggacgacttcaagggg SEQ ID NO:73 gccgtcttctacggatatacgatggacgcg SEQ ID NO:74 cagatccagctcgtccagagcgggccggagctgaagaagccgggggagagcgtgaagatctcgtgcaaggcgagcggatatacgttcacggactacgcgatgcactgggtcaagcaagcgccggggaaagggctgaagtggatggggtggatcaacacgtacacggggaagccgacatacgcggacgacttcaaggggcgattcgtgttctcgctggaggcgagcgcgagcacggcgaacctgcaaatctcgaacctgaagaacgaggacacggcgacgtacttctgcgcgcgggccgtcttctacggatatacgatggacgcgtgggggcagggtaccagcgtgacggtctcgagc SEQ ID NO: 75 cgggcgagcgaggacatctactcgaacctggcg SEQ ID NO: 76 tccgtcaagcggctgcaagac SEQ ID NO: 77 ctgcaagggagcaacttcccgctgacg SEQ ID NO: 78 gacatccagatgacgcagagcccggcgtcgctgagcgcgagcctgggggagacgatctcgatcgcgtgccgggcgagcgaggacatctactcgaacctggcgtggtatcaacagaagagcgggaagagcccgcagctgctgatcttctccgtcaagcggctgcaagacggcgtcccgagccgattctcggggagcgggagcgggacgcagtactcgctgaagatctcggggatgcagccggaggacgagggggactacttctgcctgcaagggagcaacttcccgctgacgttcgggtcgggtaccaaactcgagatcaaa SEQ ID NO: 79 tggatcaacgcgtacacggggaagccgacctacgcggacgacttcaagggg Sequence number 80 gccgtctactacggatatacgatggac Sequence number 81 cagatccagctcgtccagagcgggccggagctgaagaagccgggggagagcgtgaagatctcgtgcaagacgagcggatatacgttcacggactacgcgatgcactgggtcaagcaggggccagggaaagggatgaagtggatggggtggatcaacgcgtacacggggaagccgacctacgcggacgacttcaaggggcgattcgtgctgagcctggaggcgagcgcctcgacggcgaacctgcaaatctcgaacctgaagaacgaggacacggcgacgtacttctgcgcgcgggccgtctactacggatatacgatggacgcgtgggggcagggtaccagcgtgatcgtctcgagc Sequence number 82 cggacgagcgaggacatctactcgaacttcgcg Sequence number 83 tcagtcaaccggctgcaagac Sequence number 84 ctgcaagggagcaagttcccgctgacg Sequence number 85 gacatccagatgacgcagagcccggcgagcctgagcgcgagcctgggggagacgatctcgatcgagtgccggacgagcgaggacatctactcgaacttcgcgtggttccagcagaagagcgggaagagcccgcagctgctgatctactcagtcaaccggctgcaagacggcgtcccgagccgattctcggggagcgggagcgggacgcagtactcgctgaagatctcggggatgcagccggaggacgagggggactacttctgcctgcaagggagcaagttcccgctgacgttcgggagcggtaccaaactcgagatcaaa Sequence number 86: gactacgcgatgtac Sequence number 87: tggatcaacacgtacacggggaagccgacctacgcggacgacttcaagggg Sequence number 88 cagatccagctcgtccagagcgggccggagctgaagaagccgggggagagcgtgaagatctcgtgcaaggcgagcggatatacgttcacggactacgcgatgtactgggtcaagcaagtgccggggaaagggctgcgatggatggggtggatcaacacgtacacggggaagccgacctacgcggacgacttcaaggggcgattcgtgttctcgctggaggcgagcgcgagcacggcgaacctgcaaatctcgaacctgaagaacgaggacacggcgacgtacttctgcgcgcgggccgtcttctacggatatacgatggacgcgtgggggcagggtaccagcgtgacggtctcgagc Sequence number 89: cggacgagcgaggacatctactcgaacctggcg Sequence number 90: gcgatcaagcggctgcaagac Sequence number 91 gacatccagatgacgcagagcccggcgagcctgagcgcgagcctgggggagacgatctcgatcgcgtgccggacgagcgaggacatctactcgaacctggcgtggtatcaacagaagagcgggaagagcccgcagctgctgatcttcgcgatcaagcggctgcaagacggcgtcccgagccgattctcggggagcgggagcgggacgcagtactcgctgaagatctcggggatgcagccggaggacgagggggactacttctgcctgcaagggagcaagttcccgctgacgttcgggtcgggtaccaaactcgagatcaaa SEQ ID NO: 92 tggatcaatacatacacggggaagccgacttatgcgcaaaaattccaagga SEQ ID NO: 93 gcggtcttctacggatatacgatggatgcc SEQ ID NO: 94 caggtccaactagtccaaagcggggcggaagtcaagaagcccggagcatccgtcaaagtcagctgcaaggcgagcggatatacatttacggactacgcgatgcactgggtcaggcaagcccctgggcaaaggctcgaatggatgggatggatcaatacatacacggggaagccgacttatgcgcaaaaattccaaggaagagtcacaattacgcgggatacatccgcatctaccgcctacatggagctaagctcgctgcggagcgaggatacggcggtctactattgcgcccgagcggtcttctacggatatacgatggatgcctgggggcagggtaccctggtcacggtctcgagc SEQ ID NO: 95 agggcctccgaagacatctactccaacctggca SEQ ID NO: 96 agcgtcaaaagactacaagat SEQ ID NO: 97 ttgcaaggaagcaatttccccttgact SEQ ID NO:98 gacattcaaatgacgcaaagcccatcatcgctgagcgcatcggtcggggatagagtcaccataacatgcagggcctccgaagacatctactccaacctggcatggtatcaacaaaaaccggggaaggctccgaagctgctgatatttagcgtcaaaagac tacaagatggagtaccgagccgattttcgggaagcgggagcgggacggatttcacgctgaccatatcaagtttgcaaccggaggattttgcgacatactattgcttgcaaggaagcaatttccccttgactttcgggcaaggtaccaaggtcgagatcaaa SEQ ID NO: 99 gattatgcaatgcac SEQ ID NO: 100 tggattaacacctacacgggcaagcccacatactcccaaaaattccaagga SEQ ID NO: 101 gctgtattctatggatatacaatggatgcc SEQ ID NO:102 caggtccaattagtccaaagcggggcggaagtcaagaagccgggggcgagcgtcaaagtctcatgcaaagcgagcggatacacatttacggattatgcaatgcactgggtcaggcaagcacccggacaaaggctggaatggatgggatggattaacacctacacgggcaagcccacatactcccaaaaattccaaggaagggtcacgataacgagagacacgagcgcgagcaccggaatggatgggatggattaacacctacacgggcaagcccacatactcccaaaaattccaaggaagggtcacgataacgagagacacgagcgcgagcaccgtaccctggtcaccgtctcgagc SEQ ID NO: 103 cgaacgagcgaggacatatactcaaaccttgca SEQ ID NO: 104 gcgataaagaggctgcaagac SEQ ID NO: 105 ttgcaaggctccaaatttcccctgaca SEQ ID NO: 106 gacatccaaatgactcaaagcccatcatcgctatcggcatcggtcggggatagagtcacgataacatgccgaacgagcgaggacatatactcaaaccttgcatggtatcaacaaaagccggggaaggccccgaagctactgatattcgcgataaagaggctgcaagacggagttccatcacgattttcgggatctggctcggggaccgattttacgctgactatatcatcgctgcaaccggaagattttgcaacatactactgcttgcaaggctccaaatttcccctgacattcggacaaggtaccaaggtcgagatcaaa SEQ ID NO: 107 cggacgagcgaggatatttattcgaactttgca Sequence number 108 cagtcaatcggctacaagat Sequence number 109 gacatccaaatgacgcaatcaccgagctcgctgagcgcatctgtcggggaccgtgtcacaatcacatgccggacgagcgaggatatttattcgaactttgcatggtatcaacaaaaaccgggcaaggctccgaaacttttgatttattcagtcaatcggctacaagatggcgtcccgagccgatttagcgggagcggatcgggaaccgactttacgctgacgatatcatcgctacaaccggaggacttcgcgacttattactgcctacaagggagcaaattcccgctgacattcggacaaggtaccaaggtcgagatcaaa Sequence number 110 gattacgcaatgtac Sequence number 111 tggataaatacctatacgggaaagccaacatacgcccaaaaattccaaggc Sequence number 112 gccgtcttttatggatatacgatggacgca Sequence number 113 caggtccaactggtccaatcgggggctgaagtcaaaaagccgggggcgagcgtcaaagtcagctgcaaagcatcgggatacacatttacggattacgcaatgtactgggtcaggcaagcacccggccaacgactggaatggatgggctggataaatacctatacgggaaagccaacatacgcccaaaaattccaaggccgcgtcacaataacgcgggacacgagcgcatcgacggcttatatggaactatcatcgctgcgatcggaagacacggcggtctattattgcgcacgcgccgtcttttatggatatacgatggacgcatgggggcagggtaccctggtcacggtctcgagc SEQ ID NO: 114 gactacgcaatgcac SEQ ID NO: 115 tggattaatgcctacacggggaagccgacctacgcacaaaaattccaagga SEQ ID NO: 116 gccgtcttctatggatatacgatggatgct SEQ ID NO:117 caggtccaattggtccaaagcggggcggaggtcaagaagccgggggcgagcgtcaaagtctcatgcaaggcaagcggatatacatttacggactacgcaatgcactgggtccggcaagcccctgggcaacggctggaatggatgggatggattaatgcctacacggggaagccgacct acgcacaaaaattccaaggacgagtcacgattacgcgggatactagcgcgagcaccgcatatatggagctaagctcgctgcgatctgaggataccgctgtatactactgcgcgagagccgtcttctatggatatacgatggatgcttgggggcagggtaccctggtcacggtctcgagc SEQ ID NO: 118 cgagcttcggaggacatctatagcaacttggct SEQ ID NO: 119 agcgtcaaaaggctccaagac SEQ ID NO: 120 ctacaaggctctaacttcccattgaca SEQ ID NO:121 gatatccaaatgacgcaatcaccatctagcctatcggcctctgtgggggaccgagtcaccatcacatgccgagcttcggaggacatctatagcaacttggcttggtatcaacaaaagccggggaaagcaccaaagctgctgatatatagcgtcaaaaggc tccaagacggagtcccaagccgattctcgggctccggctccgggacggattttacgctgacaatttcgagcctgcaaccggaggactttgcaacctactattgcctacaaggctctaacttcccattgacatttgggcaaggtaccaaggtcgagatcaaa SEQ ID NO:122 gactacgctatgtat SEQ ID NO: 123 tggattaatgcctacaccgggaagccgacttatgcgcaaaaatttcaagga SEQ ID NO: 124 gcggtctactatggatatacgatggacgca SEQ ID NO:125 caggtccaactggttcaatctggagcggaagtcaagaagcccggagcatccgtcaaagtctcgtgcaaggcatctggatacacattcaccgactacgctatgtattgggtccggcaagcccccggacaacggctggaatggatgggatggattaatgcctacaccgggaagccgactt atgcgcaaaaatttcaaggaagggtcacgattacgcgggacacgagcgcctcaaccgcatacatggagctatcgagcctgcgaagcgaggacaccgcggtctactactgcgcgcgggcggtctactatggatatacgatggacgcatgggggcagggtaccctggtcacggtctcgagc Sequence number 126 WINXYTGKPTYXQKFQG The fourth X is a T or an A; The 12th X is A or S. [Only for the heavy chain CDR2 of hCl1x, This does not apply to chimeric clones cCl1-1, cCl1-2, and cCl1-3. SEQ ID NO:127 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Light chain constant region-CL domain] SEQ ID NO:128 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R [Heavy chain constant region-CH1+Fc domain] SEQ ID NO:129 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R [L234A / L235A mutations in the heavy chain constant region - CH1+Fc domain] SEQ ID NO:130 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X=K or R [L236A / L236A / P329G mutations in the heavy chain constant region - CH1+Fc domain] SEQ ID NO: 131 LPQTGG [sortase tag] SEQ ID NO: 132 GGGGS-LPQTGG [sortase tag] SEQ ID NO: 133 hCL1a heavy chain full length LALA QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X = K or R SEQ ID NO: 134 hCL1a heavy chain full length LALAPG QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMHWVRQAPGQRLEWMGWINTYTGKPTYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAVFYGYTMDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X = K or R SEQ ID NO: 135 CLDN18.2 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV SEQ ID NO:136 LPXTG m [sortase tag] The third X is any of the 20 naturally occurring amino acids, and m is 1 to 21. SEQ ID NO:137 LPXAG m [sortase tag] The third X is any of the 20 naturally occurring amino acids, and m is 1 to 21. SEQ ID NO:138 LPXSG m [sortase tag] The third X is any of the 20 naturally occurring amino acids, and m is 1 to 21. SEQ ID NO:139 LAXTG m [sortase tag] The third X is any of the 20 naturally occurring amino acids, and m is 1 to 21. SEQ ID NO:140 LPXTA m [sortase tag] The third X is any of the 20 naturally occurring amino acids, and m is 1 to 21. SEQ ID NO:141 NPQTG m [sortase tag] SEQ ID NO:142 NPQTN m [sortase tag] SEQ ID NO:143 LPETGG [sortase tag] SEQ ID NO:144 (GGGGS) o [Oligopeptide], o=1-5 SEQ ID NO: 145 LPXTGG [sortase tag] The third X is any of the 20 naturally occurring amino acids. SEQ ID NO: 146 GGGGSLPXTGG [sortase tag] The eighth X is any of the 20 naturally occurring amino acids. SEQ ID NO:147 GGGGG [oligopeptide] SEQ ID NO: 148 LPQTG [sortase tag]

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Claims

1. General formula A-(LT) ｎ or a pharma- ceutically acceptable salt or ester thereof, a. A is an antibody or fragment thereof that binds to CLDN18.2, comprising an HCDR1 sequence of SEQ ID NO: 21, an HCDR2 sequence of SEQ ID NO: 22, and an HCDR3 sequence of SEQ ID NO: 23, and an LCDR1 sequence of SEQ ID NO: 24, an LCDR2 sequence of SEQ ID NO: 25, and an LCDR3 sequence of SEQ ID NO: 26; b. L is a linker; c. T is an anthracycline or a derivative thereof as the toxic drug; n is an integer from 1 to 10; An antibody drug conjugate or a pharma- ceutically acceptable salt or ester thereof.

2. The linker comprises at least one non-cleavable linker element, preferably the non-cleavable linker element is a. Ethylenediamine (EDA), b. N-formyl-N,N'-dimethylethylenediamine, c. Diethylamine (DEA), d. the following formula: 【Chemistry 1】 Piperazine-derived compounds represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element. e. the following formula: 【Chemistry 2】 A compound represented by the formula: where the wavy lines indicate a bond to the toxic drug and a bond to another linker element. f) a compound of the formula: 【Chemistry 3】 A compound represented by the formula: (wherein the wavy line represents the bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof). g. The following formula: 【Chemistry 4】 A maleimidocaproyl compound represented by the formula: (wherein the wavy line indicates a bond to another linker element, and [Ab] indicates the antibody or fragment thereof), and h) a compound of the formula: 【Chemistry 5】 A compound represented by the formula: (In the formula, the wavy line represents the bond to the toxic drug, and [Ab] represents the antibody or a fragment thereof.) and The non-cleavable linker element is linked to the toxic drug via an amide bond or an ether bond.

2. The antibody-drug conjugate of claim 1, or a pharma- ceutically acceptable salt or ester thereof.

3. 3. The antibody-drug conjugate of claim 2, or a pharma- ceutically acceptable salt or ester thereof, wherein the linker further comprises an oligopeptide linker element, an enzymatically cleavable linker element and / or a spacer element.

4. One oligopeptide linker element contains -LPXTG as a sortase recognition motif. m -, -LPXAG m -, -LPXSG m -, -LAXTG m -, -LPXTG m -, -LPXTA m -, -NPQTG m --and--NPQTN m -, G m is an oligoglycine, where m is an integer from 1 to 21; m is oligoalanine, where m is an integer from 1 to 21; m is an oligoasparagine, where m is an integer from 1 to 21; and X is any of the possible amino acids; Preferably, the oligopeptide as a sortase recognition motif is -LPQTGG- or -LPETGG-; Preferably, the oligopeptide linker element comprises: a. the sequence of SEQ ID NO: 131; or b. Sequence SEQ ID NO: 132 4. The antibody-drug conjugate of claim 3, comprising:

5. The enzymatically cleavable linker element has the formula: 【Chemistry 6】 (wherein the wavy line indicates a bond to another linker element) or a pharma- ceutically acceptable salt or ester thereof.

6. The spacer element comprises a peptidic flexible oligopeptide, preferably the peptidic flexible oligopeptide consists of G and S, more preferably the peptidic flexible oligopeptide consists of (GGGGS) o (wherein o is 1, 2, 3, 4 or 5), or a pharma- ceutically acceptable salt or ester thereof.

7. a. The linker is an [oligopeptide linker element]-[non-cleavable linker element], preferably the linker is i. [LPXTGG]-[ethylenediamine], where X is any of the possible amino acids; and 【Chemistry 7】 where X is any of the possible amino acids; b. The linker is an oligopeptide linker element - an enzymatically cleavable linker element - a non-cleavable linker element, preferably i. [LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine], where X is any of the possible amino acids; and ii. [LPXTGG]-[vc-PAB]-[piperazine], where X is any of the possible amino acids. Selected from; c. The linker is [spacer element]-[oligopeptide linker element]-[non-cleavable linker element], preferably the linker is i. [GGGGS]-[LPXTGG]-[ethylenediamine], where X is any of the possible amino acids; and 【Chemistry 8】 where X is any of the possible amino acids; or d. The linker is: [spacer element]-[oligopeptide linker element]-[enzymatically cleavable linker element]-[non-cleavable element], preferably the linker is: i. [GGGGS]-[LPXTGG]-[vc-PAB]-[N-formyl-N,N'-dimethylethylenediamine], where X is any of the possible amino acids; and ii. [GGGGS]-[LPXTGG]-[vc-PAB]-[piperazine], where X is any of the possible amino acids. Selected from: The antibody-drug conjugate according to any one of claims 1 to 6, or a pharma- ceutically acceptable salt or ester thereof.

8. 8. The antibody-drug conjugate of claim 7, or a pharma- ceutically acceptable salt or ester thereof, wherein the non-cleavable linker element is ethylenediamine and the oligopeptide linker element is LPXTGG, where X is Q or E, preferably X is Q.

9. a. (L-T) is covalently linked to both light chains of the antibody or fragment thereof; b. (LT) is covalently linked to both heavy chains of said antibody or fragment thereof; or c. (L-T) is covalently linked to both light chains and both heavy chains of said antibody or fragment thereof; The antibody-drug conjugate according to any one of claims 1 to 8, or a pharma- ceutically acceptable salt or ester thereof.

10. The anthracycline derivative has the following formula (I): 【Chemistry 9】 (In the formula, R 1 is a hydrogen atom, a hydroxyl group or a methoxy group, R 2 is C 1 -C 5 is an alkoxy group) and wherein the non-cleavable linker element has C 13 Covalently bonded at position C 14 and the hydroxyl group is removed, or C 14 covalently linking said non-cleavable linker element at the hydroxyl group at position The antibody-drug conjugate or a pharma- ceutically acceptable salt or ester thereof according to any one of claims 1 to 9, characterized in that the anthracycline derivative is a derivative of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682).

11. The antibody or fragment thereof a. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 15, and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5, and the LCDR3 sequence of SEQ ID NO: 6; b. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 16 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; c. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 16 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 17, the LCDR2 sequence of SEQ ID NO: 14 and the LCDR3 sequence of SEQ ID NO: 11; d. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:16 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:18, the LCDR2 sequence of SEQ ID NO:19 and the LCDR3 sequence of SEQ ID NO:11; e. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 15 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; f. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5, and the LCDR3 sequence of SEQ ID NO: 6; g. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 20 and the HCDR3 sequence of SEQ ID NO: 3, and the LCDR1 sequence of SEQ ID NO: 18, the LCDR2 sequence of SEQ ID NO: 19 and the LCDR3 sequence of SEQ ID NO: 11; h. the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 20 and the HCDR3 sequence of SEQ ID NO: 8, and the LCDR1 sequence of SEQ ID NO: 4, the LCDR2 sequence of SEQ ID NO: 5 and the LCDR3 sequence of SEQ ID NO: 6; or i. HCDR1 sequence of SEQ ID NO: 12, HCDR2 sequence of SEQ ID NO: 20, and HCDR3 sequence of SEQ ID NO: 8, and LCDR1 sequence of SEQ ID NO: 17, LCDR2 sequence of SEQ ID NO: 14, and LCDR3 sequence of SEQ ID NO: 11 11. The antibody-drug conjugate of claim 1, or a pharma- ceutically acceptable salt or ester thereof, comprising:

12. The antibody or fragment thereof a. the HCDR1 sequence of SEQ ID NO:1, the HCDR2 sequence of SEQ ID NO:2 and the HCDR3 sequence of SEQ ID NO:3, and the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5 and the LCDR3 sequence of SEQ ID NO:6; b. the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 7 and the HCDR3 sequence of SEQ ID NO: 8, and the LCDR1 sequence of SEQ ID NO: 9, the LCDR2 sequence of SEQ ID NO: 10 and the LCDR3 sequence of SEQ ID NO: 11; or c. HCDR1 sequence of SEQ ID NO: 12, HCDR2 sequence of SEQ ID NO: 2, and HCDR3 sequence of SEQ ID NO: 3, and LCDR1 sequence of SEQ ID NO: 13, LCDR2 sequence of SEQ ID NO: 14, and LCDR3 sequence of SEQ ID NO: 11 Including, Preferably, the antibody or fragment thereof comprises: a. the VH sequence of SEQ ID NO: 27 and the VL sequence of SEQ ID NO: 28; b. the VH sequence of SEQ ID NO: 29 and the VL sequence of SEQ ID NO: 30; or c. The VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32 Including, The antibody-drug conjugate according to any one of claims 1 to 10, or a pharma- ceutically acceptable salt or ester thereof.

13. The antibody or fragment thereof a. the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 38; b. the VH sequence of SEQ ID NO: 34 and the VL sequence of SEQ ID NO: 38; c. the VH sequence of SEQ ID NO: 34 and the VL sequence of SEQ ID NO: 39; d. the VH sequence of SEQ ID NO: 34 and the VL sequence of SEQ ID NO: 40; e. the VH sequence of SEQ ID NO: 35 and the VL sequence of SEQ ID NO: 38; f. the VH sequence of SEQ ID NO: 36 and the VL sequence of SEQ ID NO: 41; g. the VH sequence of SEQ ID NO: 36 and the VL sequence of SEQ ID NO: 40; h. the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 41; i. the VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 38; or j. The VH sequence of SEQ ID NO: 37 and the VL sequence of SEQ ID NO: 39 Including, Preferably, the antibody or fragment thereof comprises: a. a heavy chain sequence of SEQ ID NO:46 and a light chain sequence of SEQ ID NO:51; b. a heavy chain sequence of SEQ ID NO:47 and a light chain sequence of SEQ ID NO:51; c. a heavy chain sequence of SEQ ID NO:47 and a light chain sequence of SEQ ID NO:52; d. a heavy chain sequence of SEQ ID NO:47 and a light chain sequence of SEQ ID NO:53; e. a heavy chain sequence of SEQ ID NO:48 and a light chain sequence of SEQ ID NO:51; f. the heavy chain sequence of SEQ ID NO:47 and the light chain sequence of SEQ ID NO:54; g. a heavy chain sequence of SEQ ID NO:49 and a light chain sequence of SEQ ID NO:53; h. the heavy chain sequence of SEQ ID NO:50 and the light chain sequence of SEQ ID NO:54; i. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:51; or j. a heavy chain sequence of SEQ ID NO:50 and a light chain sequence of SEQ ID NO:52; or These sequences in which the Fc domain was recombined 12. The antibody-drug conjugate of claim 1, or a pharma- ceutically acceptable salt or ester thereof, comprising:

14. A method for producing the antibody-drug conjugate or a pharma- ceutically acceptable salt or ester thereof according to any one of claims 1 to 19, comprising: a. providing as A an antibody or fragment thereof having an oligopeptide linker element in its light and / or heavy chain, preferably at its C-terminus, and optionally a spacer element between said light and / or heavy chain and said oligopeptide linker element; b. providing as T one or more toxic drugs having a non-cleavable linker element; and c. Linking the antibody or fragment thereof to the toxic drug to obtain an antibody-drug conjugate. A method comprising:

15. 1. An antibody-drug conjugate, or a pharma- ceutically acceptable salt or ester thereof, comprising: An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 46 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl groups removed. or a pharma- ceutically acceptable salt or ester thereof; An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 133 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl groups removed. or a pharma- ceutically acceptable salt or ester thereof; or An antibody that binds to CLDN18.2, which consists of two heavy chains having the amino acid sequence shown in SEQ ID NO: 134 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; a [GGGGS]-[LPQTGG]-[ethylenediamine] linker added to the C-terminus of the light chain; The linker ethylenediamine is 13 Covalently bonded at position C 14 and a small molecule toxic drug derived from anthracyclines consisting of 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682) with the hydroxyl groups removed. or a pharma- ceutically acceptable salt or ester thereof.

16. A pharmaceutical composition comprising the antibody-drug conjugate or a pharma- ceutically acceptable salt or ester thereof according to any one of claims 1 to 15, and an excipient.

17. 16. An antibody-drug conjugate or a pharma- ceutically acceptable salt or ester thereof according to any one of claims 1 to 15 for use in therapy.

18. 16. The antibody drug conjugate or a pharma- ceutically acceptable salt or ester thereof according to any one of claims 1 to 15, for use in the treatment of cancer, preferably wherein the cancer is selected from pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer and lung cancer.

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