WO2025264040A1 - Anti-claudin 18.2 antibody, anti-claudin 18.2 antibody-drug conjugate, and use thereof - Google Patents

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof binding to CLDN18.2, an antibody-drug conjugate comprising same, and a use of the antibody and the antibody-drug conjugate. An anti-CLDN18.2 monoclonal antibody according to the present invention comprises a fully human antibody sequence, thereby having low in vivo immunogenicity, and exhibits excellent antigen affinity and binding ability specific to a low expression to a high expression level of the CLDN18.2 protein. Thus, the antibody is expected to exhibit high specificity and safety as an antibody-based therapeutic agent such as in the form of a monoclonal antibody and/or an antigen-binding fragment (scFv), an antibody-drug conjugate (ADC), an immune cell engager, a chimeric antigen receptor (CAR), a multispecific antibody, and the like. In addition, the antibody according to the present invention may undergo cellular internalization, enables an anti-CLDN18.2 antibody-drug conjugate comprising said antibodies to be conveniently prepared, and has excellent yield and quality and thus is expected to be highly likely to be developed as a drug. A drug conjugate comprising the anti-CLDN18.2 antibody according to the present invention has excellent in vivo anticancer efficacy and has an expanded therapeutic index (TI) and thus is expected to be usefully employable for the treatment and/or prevention of cancer diseases expressing CLDN18.2 and related diseases.

Description

Anti-claudin 18.2 antibody, anti-claudin 18.2 antibody-drug conjugate and use thereof

The present invention relates to a novel antibody or antigen-binding fragment thereof that binds to claudin 18.2, an antibody-drug conjugate comprising the same, and a pharmaceutical composition comprising the same for the treatment of cancer.

An antibody-drug conjugate (ADC) is a compound that connects a biologically active payload (small molecule drug) to a target-directed antibody via a chemical linker. It is designed to internalize the ADC in cells expressing the target antigen on their surface, releasing the drug to induce biological activity. ADCs are innovative anticancer treatments that are more specific to tumor cells and have fewer side effects on normal cells than conventional non-specific, small molecule chemotherapy drugs. Their applications are currently expanding beyond the field of anticancer treatment to encompass other diseases.

Claudin 18.2 (Claudin 18 isoform 2, CLDN18.2) is one of the 27 isoforms belonging to the claudin family. It is known to be expressed in differentiated epithelial cells and cells in the process of cancer metastasis, while rarely expressed in normal lung and stomach epithelium. In particular, in normal tissues, it is located on the apical side of the cell, maintaining cell polarity and not exposed to the outside of the tissue. However, when cell polarity is broken during malignant transformation of gastric epithelial tissue, for example, the ECM (extracellular matrix) of claudin 18.2 is exposed to the cell surface, allowing antibody binding. Therefore, claudin 18.2 is being developed as a new cancer target for gastric cancer and other cancers (lung cancer, pancreatic cancer, etc.), and is also attracting attention as a potential target for esophageal cancer, ovarian cancer, etc.

Currently, the development of single antibodies and antibody-drug conjugates targeting claudin 18.2 has been reported, but there is still a need for the development of anti-claudin 18.2 antibodies and antibody-drug conjugates with higher efficacy and safety (US Patent Publication No. 2018-0117174 A1).

Accordingly, the inventors of the present invention have endeavored to develop antibodies and antibody-drug conjugates with higher efficacy and safety, and have completed the present invention by confirming that the anti-CLDN18.2 antibody-drug conjugate according to the present invention exhibits excellent anticancer activity.

In order to achieve the above object, one aspect of the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

Another aspect of the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment, a vector comprising the polynucleotide, and a cell transformed with the vector.

Another aspect of the present invention provides a method for producing an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising the steps of: i) culturing the transformed cell; and ii) obtaining the antibody or antigen-binding fragment thereof from the culture medium of the cell.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

Another aspect of the present invention is a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 31; a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 6; Or, a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 8; and an antibody-drug conjugate comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; and an anticancer agent.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate as an active ingredient.

Another aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate, for preventing or treating cancer.

Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject the antibody or antigen-binding fragment thereof, or antibody-drug conjugate.

The novel anti-CLDN18.2 monoclonal antibody according to the present invention is composed of a fully human antibody sequence and has low immunogenicity in the body. In addition, it exhibits excellent antigen affinity and specific binding ability to the CLDN18.2 protein. Therefore, the antibody is expected to exhibit high specificity and safety as an antibody-based therapeutic agent in the form of a monoclonal antibody and/or an antigen-binding fragment (single chain fragment variable, scFv), an antibody-drug conjugate (ADC), an immune cell engager (T-cell or NK-cell engager, Engager), a chimeric antigen receptor (CAR), a multispecific antibody, etc. In addition, the antibody according to the present invention is capable of cell internalization, and the production of an anti-CLDN18.2 antibody-drug conjugate containing the antibody is convenient and has excellent yield and quality, so it is expected to have high potential for development as a drug. The drug conjugate comprising the anti-CLDN18.2 antibody according to the present invention is expected to have excellent in vivo anticancer efficacy and an expanded therapeutic index (TI), and thus can be usefully utilized in the treatment and/or prevention of cancer diseases expressing CLDN18.2 and related diseases.

Figure 1 shows the SDS-PAGE results of purified anti-CLDN18.2 antibody.

Figures 2a and 2b show the results of SEC (Size exclusion chromatography)-UPLC (Ultra performance liquid chromatography) analyzing the purity of purified anti-CLDN18.2 antibody.

Figures 3a and 3b are the results of HIC (Hydrophobic interaction chromatography)-UPLC analyzing the hydrophilicity of purified anti-CLDN18.2 antibody.

Figure 4 shows the results of Differential Scanning Fluorimetry (DSF) analyzing the thermal stability of purified anti-CLDN18.2 antibody.

Figure 5 shows the results of FACS analysis confirming the binding affinity of anti-CLDN18.2 antibodies to CLDN18.2 at the cellular level. Figure 5a shows the results confirmed in HEK293E cells, a CLDN18.2-negative cell line, and Figure 5b shows the results confirmed in CLDN18.2/HEK293E cells, a CLDN18.2-overexpressing cell line. Figure 5c shows the results confirmed in SNU-601 cells, a CLDN18.2-positive human gastric cancer cell line, Figure 5d shows the results confirmed in PATU8988s cells, a CLDN18.2-positive human pancreatic cancer cell line, and Figure 5e shows the results confirmed in BxPC3 cells, a CLDN18.2-negative and CLDN18.1-positive human pancreatic cancer cell line.

Figure 6 shows the results of confocal microscopy confirming the cellular internalization of anti-CLDN18.2 antibodies. Figure 6a shows the cellular internalization of IgG, and Figure 6b shows the cellular internalization of TAB01 antibodies. Figure 6c shows the cellular internalization of TAB07 antibodies, and Figure 6d shows the cellular internalization of TAB10 antibodies.

Figure 7 shows the drug-antibody conjugation rate of anti-CLDN18.2 antibody-drug conjugate The results are as follows: The DAR (Density-Analysis-Ratio) was confirmed through RP (Reversed-phase chromatography)-UPLC or rRP-UPLC (Reduced reverse phase-UPLC). Figures 7a to 7c show the results analyzed by RP-UPLC, and Figures 7c to 7k show the results analyzed by rRP-UPLC.

Figure 8 shows the results of analyzing the purity of an anti-CLDN18.2 antibody-drug conjugate using SEC-UPLC. Figures 8a and 8b show the results of an antibody-drug conjugate in which an anti-CLDN18.2 antibody and MMAE, MMAF, or Exatecan are conjugated via a linker, which is an embodiment of the present invention, and Figures 8c and 8d show the results of analyzing the purity of an antibody-drug conjugate in which an anti-CLDN18.2 antibody and MMAE, Belotecan, or Exatecan are conjugated via a linker, which is an embodiment of the present invention. Figures 8e and 8f show the results of analyzing the purity of antibody-drug conjugates conjugated with anti-CLDN18.2 antibodies and clinically proven linker-payloads (VC-PAB-MMAE, GGFG-DXD, mc-MMAF), and Figure 8g shows the results of analyzing the purity of antibody-drug conjugates conjugated with anti-CLDN18.2 antibodies and Exatecan via a linker, which is an embodiment of the present invention.

Figure 9 is a graph showing the results of comparing the hydrophilicity of an antibody-drug conjugate (TAB07.409.1) in which an anti-CLDN18.2 antibody and Exatecan are conjugated via a linker, which is an example of the present invention, and TAB07.121.1 (GGFG-DXD) in which an anti-CLDN18.2 antibody and a clinically proven Enhertu linker are introduced.

Figure 10 shows the results of confirming the cytotoxicity of the anti-CLDN18.2 antibody-drug conjugate at the cellular level. Figures 10a and 10b show the results of confirming the cytotoxicity of the anti-CLDN18.2 antibody-drug conjugate conjugated with four drugs in a specific antibody position according to each cell line. Figures 10c and 10d show the results of confirming the cytotoxicity of the anti-CLDN18.2 antibody-drug conjugate conjugated with two drugs in a human gastric cancer cell line (SNU-601) and a CLDN18.2-overexpressing human kidney cell line (CLDN18.2/HEK293E), respectively. Figures 10e and 10f show the results of confirming the cytotoxicity of an anti-CLDN18.2 antibody-drug conjugate conjugated with four drugs in a CLDN18.2-positive human cell line (CLDN18.2/HEK293E, SNU-601) and a human pancreatic cancer cell line (PATU8988s), respectively, and Figure 10g shows the results of confirming the cytotoxicity of an anti-CLDN18.2 antibody-drug conjugate conjugated with five or more drugs in a human gastric cancer cell line (SNU-601). Figure 10h shows the results of confirming the cytotoxicity of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is an example of the present invention, in a cell line (-CLDN18.2, CHO-K1) in which CLDN18.2 is not overexpressed on the cell surface (Negative) and a CLDN18.2-overexpressing human kidney cell line (CLDN18.2/HEK293E).

Figure 11 shows the results of confirming the anticancer efficacy of an anti-CLDN18.2 antibody-drug conjugate in a xenograft tumor model mouse transplanted with a CLDN18.2-positive human cancer cell line. Figure 11a shows the results of confirming the anticancer efficacy of an anti-CLDN18.2 antibody-drug conjugate in a subcutaneous xenograft tumor model (SUN-601 subcutaneous xenograft model) of a human gastric cancer cell line (SUN-601). Figure 11b shows the results of confirming the anticancer efficacy of an anti-CLDN18.2 antibody-drug conjugate in a subcutaneous xenograft tumor model (PATU8988s subcutaneous xenograft model) of a human pancreatic cancer cell line (PATU8988s). Figure 11c is a graph showing the results of comparing the anticancer effects of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is an example of the present invention, and an antibody-drug conjugate (TAB07.121.1, GGFG-DXD) in which an anti-CLDN18.2 antibody and a clinically proven Enhertu linker are introduced, in a tumor mouse model implanted with a human gastric cancer cell line (SNU-601 cells).

Figure 12 shows the results of confirming the stability of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is one specific example of the present invention, in rat plasma and human plasma.

Figure 13 is a graph showing the results of confirming the drug antibody ratio (DAR) of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is one specific example of the present invention, in rat plasma and human plasma.

Figure 14 is a graph showing the results of confirming pharmacokinetics after a single intravenous administration of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is one specific example of the present invention, to a rat.

anti-CLDN18.2 antibody

As used herein, the term "Claudin18.2 (CLDN18.2)" refers to one of the subtypes of claudin (CLDN). Claudin is an important component of tight junctions that form the cell-peripheral barrier. CLDN is expressed in various tissues, such as gastric, pancreatic, and lung tissues, and is associated with the formation of cancer. CLDN18.2 is a highly selective marker protein expressed only in differentiated gastric mucosal epithelial cells, and its expression is very limited in normal, healthy tissues and is known to not be expressed in undifferentiated gastric stem cells. In addition, CLDN18.2 has been reported to be overexpressed in pancreatic cancer, esophageal cancer, ovarian adenocarcinoma, and lung cancer.

In the present invention, the CLDN18.2 protein may be included without limitation as long as it is derived from mammals, including primates such as humans and monkeys, and rodents such as rats and mice.

In addition, the CLDN18.2 protein may include, but is not limited to, both a native or mutant CLDN18.2 protein. The native CLDN18.2 protein generally refers to a polypeptide comprising the amino acid sequence of the native CLDN18.2 protein. The amino acid sequence and polynucleotide sequence for the CLDN18.2 protein can be obtained from known databases such as the GenBank of the National Center for Biotechnology Information (NCBI) in the United States.

The term "anti-CLDN18.2 antibody" as used herein means an antibody capable of binding to CLDN18.2, and may be used interchangeably herein with "antibody specific for CLDN18.2" or "antibody that specifically binds to CLDN18.2."

The above “antigen-binding fragment” means a fragment having an antigen-binding function.

Specifically, the antibody and antigen-binding fragment may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a single domain antibody, a single chain antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, an intrabody, an Fv, a scFv, an Fv (di-scFv) linked by a disulfide bond, a Fab fragment, an F(ab') ₂ fragment, and an epitope-binding fragment of any of the above.

As used herein, the term "epitope" refers to an antigenic determinant, a region on an antigen to which an antibody or polypeptide binds. A protein epitope may include amino acid residues directly involved in binding, as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide. It is the simplest form or smallest structural region of a complex antigen molecule capable of binding to an antibody or receptor. An epitope may be linear or structural/conformational.

In the present invention, the antibody comprises a heavy chain and a light chain, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain and light chain may comprise a constant region and a variable region.

As used herein, the term "heavy chain (HC)" is meant to include both full-length heavy chains and fragments thereof, which include a variable region (VH) sufficient to confer specificity for an antigen and three constant regions, CH1, CH2 and CH3.

As used herein, the term "light chain (LC)" is meant to include both full-length light chains and fragments thereof that include a variable region (VL) and a constant region (CL) sufficient to confer specificity for an antigen.

The light and heavy chain variable regions of the antibody comprise three hypervariable regions, called complementarity determining regions (CDRs), and four framework regions (FRs). The CDRs primarily bind to epitopes on the antigen. The CDRs of each chain are typically sequentially named CDR1, CDR2, and CDR3, starting from the N-terminus, and are identified by the chain on which the specific CDR is located.

In the present invention, the anti-CLDN18.2 antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 6. In this case, the heavy chain variable region may include an amino acid sequence of SEQ ID NO: 9, and the light chain variable region may include an amino acid sequence of SEQ ID NO: 10.

In addition, the anti-CLDN18.2 antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In this case, the heavy chain variable region may include the amino acid sequence of SEQ ID NO: 9, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 11.

The heavy chain variable region of the antibody may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or 100% identity with each amino acid sequence of SEQ ID NO: 9. In addition, the light chain variable region of the antibody may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or 100% identity with each amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

The heavy chain constant regions (CH) of immunoglobulins exhibit different amino acid compositions and sequences, and thus possess different types of antigenicity. Therefore, immunoglobulins can be classified into five categories and referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Furthermore, the same type of Ig can be classified into different subtypes based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain can be classified as κ or λ chain depending on the different constant regions. Each of the five types of IgG can have κ or λ chains. Here, immunoglobulin refers to a glycoprotein that functions as an antibody, in the same sense as an antibody.

When the anti-CLDN18.2 antibody of the present invention comprises a heavy chain constant region, it may comprise a constant region derived from IgG, IgA, IgD, IgE, IgM, or a partial mixture (hybrid) thereof. In one specific example, the constant region may be derived from IgG1, and specifically may comprise the amino acid sequence of SEQ ID NO: 32.

As used herein, the term "hybrid" means that within a single-chain immunoglobulin heavy chain constant region, there are sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins. For example, a hybrid domain consisting of one to four domains selected from the group consisting of CH1, CH2, and CH3 of IgG, IgA, IgD, IgE, and IgM is possible.

Additionally, when the anti-CLDN18.2 antibody comprises a light chain constant region (LC), the light chain constant region may be derived from a κ or λ light chain. In one specific example, the light chain constant region may be derived from a κ, and specifically, may comprise the amino acid sequence of SEQ ID NO: 33.

In the present invention, the constant region of the antibody may include a mutation.

Specifically, the heavy chain constant region of the antibody may have a different glycosylation pattern from that of the wild-type heavy chain constant region, or may have increased, decreased, or deglycosylated glycosylation compared to the wild-type heavy chain constant region. In addition, an aglycosylated heavy chain constant region is also included. The heavy chain constant region or a variant thereof may have a controlled number of sialic acid, fucosylation, glycosylation, etc. through culture conditions or genetic manipulation of the host. In addition, the glycosylation of the heavy chain constant region of the immunoglobulin may be modified by a conventional method, such as a chemical method, an enzymatic method, or a genetic engineering method using a microorganism. In addition, the heavy chain constant region variant may be a mixed form of the Fc region of the immunoglobulin IgG, IgA, IgE, IgD, or IgM. Additionally, the heavy chain constant region variant may be a form in which some amino acids of the heavy chain constant region are substituted. Specifically, it may be a form in which some amino acids of the CH2 region are substituted.

The “amino acid” introduced by the above substitution and/or addition may be any one selected from the group consisting of lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

In one embodiment, the mutation in the heavy chain constant region may be a substitution of an amino acid in the CH2 region of the heavy chain constant region with L234A/L235A (LALA). Specifically, it may include the amino acid sequence of SEQ ID NO: 14.

Additionally, the antibody light chain constant region may contain mutations.

Specifically, the mutation in the light chain constant region may be a substitution of the amino acid in the constant region derived from the κ light chain with K149C. Specifically, it may include the amino acid sequence of SEQ ID NO: 15.

Polynucleotide encoding anti-CLDN18.2 antibody

Another aspect of the present invention provides a polynucleotide encoding the anti-CLDN18.2 antibody or an antigen-binding fragment thereof. Specifically, the polynucleotide encoding the heavy chain variable region of the antibody or an antigen-binding fragment thereof may comprise the nucleic acid sequence of SEQ ID NO: 34. In addition, the polynucleotide encoding the light chain variable region of the antibody or an antigen-binding fragment thereof may comprise the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 28.

The polynucleotide may have one or more bases substituted if it encodes the same polypeptide. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

Specifically, the polynucleotide may comprise a base sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to each of the nucleic acid sequences of SEQ ID NO: 34, SEQ ID NO: 20, and SEQ ID NO: 28.

The polynucleotide may additionally comprise a nucleic acid encoding a signal sequence or a leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that directs the secretion of a target protein. The signal peptide is cleaved after being translated in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates the movement of a protein across the endoplasmic reticulum (ER) membrane. The signal sequence is well known in the art and typically comprises 16 to 30 amino acid residues, but may comprise more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region comprises 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during the movement of the immature polypeptide. After initiation, the signal sequence is cleaved within the lumen of the ER by a cellular enzyme commonly known as a signal peptidase. At this time, the signal sequence may be a secretion signal sequence of tPa (Tissue Plasminogen Activation), HSV gDs (Signal sequence of Herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells including mammals can be used. In addition, the signal sequence may be a wild-type signal sequence or may be used by substituting a codon with a high expression frequency in the host cell.

A vector containing a polynucleotide

Another aspect of the present invention provides a vector comprising a polynucleotide encoding the anti-CLDN18.2 antibody or an antigen-binding fragment thereof. Specifically, the heavy chain may comprise the polynucleotide of SEQ ID NO: 26, and the light chain may comprise the polynucleotide of SEQ ID NO: 29 or SEQ ID NO: 30.

The above vector may be two vectors each containing a nucleic acid sequence encoding the heavy chain and the light chain, or a bicistronic expression vector containing both of the above nucleic acid sequences.

As used herein, the term "vector" refers to a nucleic acid vector that can be introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle comprising a nucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, minichromosomes, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

Specifically, the vector may be a plasmid DNA, phage DNA, etc., and may include commercially developed plasmids (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.), phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (e.g., retrovirus, adenovirus, vaccinia virus, etc.), insect virus vectors (e.g., baculovirus, etc.), etc. It can be. Since the protein expression amount and formula of the above vector differ depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

Additionally, the plasmid may contain a selection marker, such as an antibiotic resistance gene, and the host cell harboring the plasmid may be cultured under selective conditions.

As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an antibody or antigen-binding fragment thereof. A useful expression vector may be RcCMV (Invitrogen) or a variant thereof. The expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the gene of interest in mammalian cells and a bovine growth hormone (BOH) polyadenylation signal sequence to increase the steady-state level of post-transcriptional RNA.

Transformed cells expressing anti-CLDN18.2 antibodies

Another aspect of the present invention provides a transformed cell into which a vector comprising a polynucleotide encoding the anti-CLDN18.2 antibody or an antigen-binding fragment thereof has been introduced.

As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. The transformed cell can be produced by introducing the vector into a host cell and transforming the cell. Furthermore, the polynucleotide contained in the vector can be expressed to produce the anti-CLDN18.2 antibody of the present invention or an antigen-binding fragment thereof.

The above transformation can be performed by various methods and is not particularly limited thereto as long as the anti-CLDN18.2 antibody of the present invention or an antigen-binding fragment thereof can be produced. Specifically, the transformation method may include CaCl ₂ precipitation, the Hanahan method which increases efficiency by using a reducing substance called DMSO (Dimethyl sulfoxide) in the CaCl ₂ precipitation, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation/inhibition-mediated transformation. In addition, the target object can be delivered into the cell using a virus particle by infection. In addition, the vector can be introduced into the host cell by gene bombardment, etc.

In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention or an antigen-binding fragment thereof. Specifically, the host cell may include, but is not limited to, a prokaryotic cell, a eukaryotic cell, a mammal, a plant, an insect, a fungus, or a cell of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2/0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.

To optimize the therapeutic properties of the anti-CLDN18.2 antibody or antigen-binding fragment thereof or for other purposes, the glycosylation-related genes of the host cell can be manipulated using a method known to those skilled in the art to adjust the sugar chain pattern (e.g., sialic acid, fucosylation, glycosylation) of the anti-CLDN18.2 antibody or antigen-binding fragment thereof.

Method for producing anti-CLDN18.2 antibodies

Another aspect of the present invention provides a method for preparing the anti-CLDN18.2 antibody or antigen-binding fragment thereof.

The method for producing the anti-CLDN18.2 antibody or antigen-binding fragment thereof may include the steps of i) culturing the transformed cell; and ii) obtaining the anti-CLDN18.2 antibody or antigen-binding fragment thereof from the cell culture. Here, the transformed cell is as described above.

The method for culturing the above-mentioned transformed cells can be performed using methods widely known in the art. The culturing is not particularly limited as long as it can express and produce the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention. Specifically, the culturing can be performed continuously in a batch process, fed batch process, or repeated fed batch process (Fed Batch or Repeated Fed Batch process), but is not limited thereto.

In addition, the step of obtaining the anti-CLDN18.2 antibody or antigen-binding fragment thereof from the culture can be performed by a method known in the art. Specifically, the obtaining method is not particularly limited as long as it can obtain the produced anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention. Preferably, the obtaining method can be a method such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion).

Pharmaceutical composition comprising anti-CLDN18.2 antibody

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-CLDN18.2 antibody or an antigen-binding fragment thereof as an active ingredient. Here, the anti-CLDN18.2 antibody or an antigen-binding fragment thereof is as described above.

The term "cancer" as used herein refers to a disease caused by cells that have aggressive characteristics in which cells divide and proliferate while ignoring normal growth limits, invasive characteristics in which cells invade surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body, and is used with the same meaning as malignant tumor.

The cancer may be selected from the group consisting of, but is not limited to, stomach cancer, liver cancer, lung cancer, non-small cell lung cancer, colon cancer, bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, larynx cancer, esophagus cancer, pancreatic cancer, tongue cancer, skin cancer, bladder cancer, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, anal cancer, colon cancer, and central nervous system tumor.

In addition, "treatment of cancer" means inhibiting or preventing the growth of cancer cells or tissues, and this also includes reducing the growth and metastasis of cancer compared to when no treatment or treatment is performed, and reducing resistance to anticancer drugs so that the treatment effect is more effective. The cancer metastasis refers to the process in which tumor (cancer) cells spread to distant parts of the body, and "resistance to anticancer drugs" or "anticancer drug resistance" refers to the absence of a therapeutic effect from the beginning of treatment when treating a cancer patient using an anticancer drug, or the cancer treatment effect is lost during the course of continued treatment although the cancer treatment effect is initially effective. "Prevention" refers to all acts of inhibiting the occurrence of cancer or delaying its onset by administering the pharmaceutical composition.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the anti-CLDN18.2 antibody or antigen-binding fragment thereof may be included in any amount (effective amount) depending on the intended use, formulation, compounding purpose, etc., as long as it can exhibit anticancer activity. Here, the "effective amount" refers to the amount of an effective ingredient capable of inducing an anticancer effect. Such an effective amount can be experimentally determined within the ordinary ability of a person skilled in the art. The pharmaceutical composition of the present invention may contain the antibody or antigen-binding fragment thereof as an effective ingredient in an amount of about 0.1 wt% to about 90 wt%, specifically about 0.5 wt% to about 75 wt%, and more specifically about 1 wt% to about 50 wt%, based on the total weight of the composition.

Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced efficacy, and can be measured by comparing parameters such as clearance rate and the treatment or improvement of cancer in test animals or human subjects.

The pharmaceutical composition of the present invention may include a conventional, non-toxic, pharmaceutically acceptable carrier that is formulated into a formulation according to a conventional method.

The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as sweeteners, solubilizers, wetting agents, emulsifiers, isotonic agents, absorbents, antioxidants, preservatives, lubricants, fillers, buffers, and bacteriostatic agents may be added.

The composition of the present invention can be prepared in various dosage forms for parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection). When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier and a method known in the art. Injectable preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin. Meanwhile, injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

Formulation of pharmaceutical compositions is well known in the art, and reference can be made to references such as Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

The antibody or composition of the present invention may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

The term "administration" as used herein refers to introducing a given substance into a subject in an appropriate manner, and the route of administration of the composition may be any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and rectal administration.

Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, the therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

Specifically, the dosage of the composition of the present invention may vary depending on the patient's age, sex, and weight, and is generally administered at about 0.001 mg to about 1,000 mg per kg of body weight, or about 0.05 mg to about 200 mg per kg, daily or every other day, or divided into one to three doses per day. However, the dosage may vary depending on the route of administration, severity of the disease, sex, body weight, age, etc., and thus the scope of the present invention is not limited thereto.

The subjects to which the above pharmaceutical composition can be applied (prescribed) are mammals and humans, and humans are particularly preferred.

The antibody of the present invention or a pharmaceutical composition comprising the same may be administered as an individual therapeutic agent or in combination with another therapeutic agent, may be administered sequentially or simultaneously with conventional therapeutic agents, or may be administered singly or in multiple doses. In this case, the other therapeutic agent may additionally include any compound or natural extract known to have proven safety and anticancer activity to enhance or enhance anticancer activity. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum effect with minimal or no side effects, at a minimum amount. This can be readily determined by those skilled in the art.

Another aspect of the present invention provides a use of an anti-CLDN18.2 antibody or antigen-binding fragment thereof for preventing or treating cancer.

Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject the anti-CLDN18.2 antibody or antigen-binding fragment thereof. The anti-CLDN18.2 antibody or antigen-binding fragment thereof, administration, cancer, prevention, and treatment are as described above.

The subject may be a mammal, preferably a human. Additionally, the subject may be a patient suffering from cancer or a subject at high risk of suffering from cancer.

The above-mentioned antibody may be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and administration frequency can be selected within an appropriate range by a person skilled in the art. A preferred dosage of the anti-CLDN18.2 antibody or antigen-binding fragment thereof may be about 0.1 mg to about 1,000 mg per kg of body weight, or about 5 mg to about 200 mg per kg of body weight, administered daily or every other day, or divided into one to three times a day, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Such dosage should not be construed as limiting the scope of the present invention in any way.

Additionally, the anti-CLDN18.2 antibody or antigen-binding fragment thereof may be administered in combination with any compound or natural extract known to have a cancer therapeutic effect, or may be formulated in the form of a combination preparation with other drugs.

antibody-drug conjugate

Another aspect of the present invention provides an antibody-drug conjugate comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; and an anticancer agent.

anti-CLDN18.2 antibody

At this time, the anti-CLDN18.2 antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 31. At this time, the heavy chain variable region may include an amino acid sequence of SEQ ID NO: 9, and the light chain variable region may include an amino acid sequence of SEQ ID NO: 13.

The anti-CLDN18.2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 6. In this case, the heavy chain variable region may comprise an amino acid sequence of SEQ ID NO: 9, and the light chain variable region may comprise an amino acid sequence of SEQ ID NO: 10.

The anti-CLDN18.2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In this case, the heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 9, and the light chain variable region may comprise the amino acid sequence of SEQ ID NO: 11.

The antibody and antigen binding fragments are the same as described above.

As used herein, the term "antibody-drug conjugate (ADC)" refers to a therapeutic agent that exhibits high anticancer efficacy by chemically combining an antibody and a drug. The antibody and drug may be covalently linked via a linker.

Specifically, the antibody-drug conjugate may have a structure of structural formula I or structural formula II:

<Structural formula I>

Ab-[L-D]n

<Structural Formula II>

Ab-[L'-D ₂ ] _n

In the above structural formulas I and II,

The above Ab is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof,

L and L' are each independently a linker or a direct bond,

The above D is an anticancer agent,

The above n can be a real number from 1 to 10. In one embodiment, n can be a real number from 1 to 8, a real number from 1 to 6, or a real number from 1 to 4. Specifically, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

D may have the structure of a residue that forms a linkage between the anticancer drug molecule and a linker or antibody. For example, D may refer to an amine radical in which a hydrogen radical is removed from a primary amine group or a secondary amine group in the anticancer drug molecule. For example, D may refer to an amine radical in which a hydrogen radical is removed from a primary amine group of belotecan, or a radical in which a hydrogen radical is removed from a secondary amine group of MMAF, exatecan, and deruxtecan.

In structural formula II, L'-D ₂ is a residue containing two drug molecule structures, which may mean a structure in which linker L' is divided into two branches and conjugated to two anticancer drug molecules D, or a structure in which two anticancer drug molecules 2 are directly conjugated to an antibody.

At this time, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is as described above.

anticancer drugs

In the antibody-drug conjugate of the present invention, the drug may be an anticancer agent.

The above "anticancer agent" collectively refers to all drugs effective in treating cancer. In the present invention, the anticancer agent may include any anticancer agent that can be used as the payload of an antibody-drug conjugate. Specifically, the anticancer drugs include Methotrexate, Taxol, L-asparaginase, Mercaptopurine, Thioguanine, Hydroxyurea, Cytarabine, Cyclophosphamide, Ifosfamide, Nitrosourea, Cisplatin, Carboplatin, Mitomycin, Dacarbazine, Procarbazine, Topotecan, Nitrogen mustard, Cytoxan, Etoposide, 5-fluorouracil, and Monomethylauristatin. Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Bis-chloroethylnitrosourea (BCNU), Irinotecan, Camptothecin, Exatecan, Blotecan, Deruxtecan, Bleomycin, Doxorubicin, Idarubicin, Daunorubicin, Dactinomycin, Plicamycin, Mitoxantrone, Asparaginase, Vinblastine, Vincristine, Vinorelbine, Paclitaxel, Docetaxel, Chlorambucil, Melphalan, Carmustine, Lomustine, Busulfan, Treosulfan, Decarbazine, Etoposide, Teniposide, Topotecan, 9-aminocamptothecin, Crisnatol, Mitomycin C, Trimetrexate, Mycophenolic acid, Tiazofurin, Ribavirin, EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), Hydroxyurea, Defoxamine, Fluxuridine, Doxifluridine, Raltitrexed, Cytarabine (ara C), Cytosine arabinoside, Fludarabine, Tamoxifen, Raloxifene, Megestrol, Goserelin, Leuprolide acetate, Flutamide, Bicalutamide, EB1089, CB1093, KH1060, Verteporfin, Phthalocyanine, Photosensitizer Pe4, Demethoxy-hypocrelin Demethoxy-hypocrellin A, Interferon-α, Interferon-γ, tumor necrosis factor, Gemcitabine, Velcade, Revamid, Thalamid, Lovastatin, 1-methyl-4-phenylpyridinium ion, Staurosporine, Actinomycin D, Dactinomycin, Bleomycin A2, Bleomycin B2, Peplomycin, Epirubicin, Prarubicin, Zorubicin, It may be one or more selected from the group consisting of, but is not limited to, Mitoxantrone, Verapamil and Thapsigargin.

In one embodiment of the present invention, the anticancer agent may be monomethyloristatin E (MMAE), monomethyloristatin F (MMAF), exatecan, belotecan, or deruxtecan.

The above monomethyl auristatin E (MMAE) refers to (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide. In this case, the above monomethyl auristatin E (MMAE) may include a structure represented by the following chemical formula I or a pharmaceutically acceptable salt thereof:

<Chemical Formula I>

.

The above monomethyloristatin F (MMAF) refers to (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid. In this case, the above monomethyloristatin F (MMAF) may include a structure represented by the following chemical formula II or a pharmaceutically acceptable salt thereof:

<Chemical Formula II>

.

The above exatecan refers to (1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione. In this case, the exatecan may include a structure of the following chemical formula III or a pharmaceutically acceptable salt thereof:

<Chemical Formula III>

.

The above belotecan refers to (4S)-4-Ethyl-4-hydroxy-11-[2-(isopropylamino)ethyl]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione. In this case, the above belotecan may include a structure of the following chemical formula IV or a pharmaceutically acceptable salt thereof:

<Chemical Formula IV>

.

The deruxtecan is 6-(2,5-dioxopyrrol-1-yl)-N-[2-[[2-[[(2S)-1-[[2-[[2-[[(10S,23S)-10-ethyl-18-fluoro-10-h ydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]t etracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]amino]-2-oxoethoxy]methylamino]-2-oxoethyl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-2-oxoethyl]amino]-2-oxoethyl]hexanamide It means. At this time, the above-mentioned deruxtecan may include a structure of the following chemical formula IIIA or a pharmaceutically acceptable salt thereof:

<Chemical Formula IIIA>

.

Linker

In the present invention, the antibody or antigen-binding fragment thereof and the anticancer agent may be linked via a linker.

As used herein, the term "linker" refers to a component of an antibody-drug conjugate that connects an antibody or an antigen-binding fragment thereof to a drug (or payload) via a chemical bond. The linker can covalently bind the antibody and the drug.

At this time, the linker (or linker-drug) may be randomly conjugated via lysine (K) of the antibody, or may be non-specifically conjugated to the antibody via cysteine (C) exposed when the disulfide bond chain is reduced. Alternatively, the linker (or linker-drug) may be induced to be conjugated to a specific position of the antibody by genetically engineering an amino acid residue at a specific position of the antibody or artificially adding an amino acid sequence to the antibody.

In one embodiment of the present invention, a linker (or linker-drug) can be conjugated non-specifically to an antibody position by reducing an antibody molecule by adding a reducing agent and then treating the antibody with a linker-drug at a molar concentration of at least four times that of the antibody. In one embodiment of the present invention, a linker (or linker-drug) can be specifically conjugated to a specific position of the antibody by specifically binding to an anti-CLDN18.2 antibody (Kappa LC, K149C) substituted with a thiol group at a specific position of the antibody.

The above linker may be a cleavable linker or a non-cleavable linker.

The term "non-cleavable linker" as used herein refers to a linker from which the drug is released when the antibody-drug conjugate is internalized into a target cell and then undergoes catabolism by cytoplasmic or lysosomal hydrolases.

In one specific example, the non-cleavable linker may be a maleimide linker. The maleimide linker may be, for example, a maleimidocaproyl (MC) linker or a succimidyl 4-(N-aleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

The term "cleavable linker" as used herein refers to a linker in the antibody-drug conjugate from which the drug is released by cleavage of the linker. The cleavable linker can be classified according to the principle by which cleavage occurs.

The above linker may be chemically cleavable or enzymatically cleavable.

In one specific example, the chemically cleavable linker may be an acid-labile linker or a reducible linker.

The term "acid-labile linker" as used herein refers to a linker that is stable in a neutral pH, such as blood, but undergoes cleavage by acid hydrolysis in an acidic environment (pH 5 to 6), such as a cancer cell microenvironment, lysosome, or endosome. The acid-labile linker includes a hydrazone linker or an ester linker.

As used herein, the term "reducible linker" refers to a linker that undergoes cleavage upon reduction by a reducing agent. In one specific example, the reducible linker may be a disulfide linker.

As used herein, the term "enzyme-cleavable linker" refers to a linker that is cleaved by a reducing agent within a cell. The enzyme-cleavable linker may be a peptide-based linker or a specific enzyme-based linker.

As used herein, the term "peptide-based linker" refers to a linker that is cleaved by an enzyme that is relatively abundant within a cell, and that includes a specific peptide bond cleavage site. The peptide-based linker may be a linker that includes one or more binding sites selected from the group consisting of valine-citrulline, valine-alanine, and phenylalanine-glycine. In one specific example, the peptide-based linker may be, but is not limited to, a valine-citrulline linker, a valine-alanine linker, an alanine-alanine-alanine, or a phenylalanine-glycine linker.

As used herein, the term "enzyme-specific linker" refers to a linker designed to be cleaved by a specific enzyme. The enzyme-specific linker may be cleaved by one or more enzymes selected from the group consisting of β-galactoside, β-glucuronide, and phosphodiester. In one embodiment, the enzyme-specific linker may be selected from the group consisting of β-galactoside linkers, β-glucuronide linkers, phosphodiester linkers, and combinations thereof.

In one specific example, the second type non-cleavable linker may be, but is not limited to, a Maleimidocaproyl (MC) linker or a Succimidyl 4-(N-aleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

In one specific example, the cleavable linker may be any one selected from the group consisting of a hydrazone linker, an ester linker, a disulfide linker, a valine-citrulline linker, a valine-alanine linker, an alanine-alanin-alanin linker, a phenylalanine-glycine linker, a β-galactoside linker, a β-glucuronide linker, a phosphodiester linker, and combinations thereof.

In one specific example, the linker may include a structural modification. In one specific example, the linker may have polyethylene glycol (PGE) substituted in a portion of the structure, or may include a self-immolative group (SIG). The "self-immolative group" refers to a site interposed between the linker and the drug, connecting the drug and the linker, and being hydrolyzed by an enzymatic reaction to dissociate the drug.

Specific examples of antibody-drug conjugates

In the present invention, the antibody-drug conjugate may be one in which one or more anticancer agents are conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof via one to ten linkers. At this time, the linker may include polyethylene glycol (PEG) and a self-immolative group (β-galactoside or β-galacturonide). More specifically, the antibody-drug conjugate may be one in which one, two, three, four, five, six, seven, eight, nine, or ten anticancer agents are conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof via one, two, three, four, five, six, seven, eight, nine, or ten linkers.

In one specific example, the molecular ratio of the linker and the drug in the antibody-drug conjugate may be about 1:1 to 1:10. For example, the molecular ratio of the linker and the drug may be about 1:1 to 1:8, about 1:1 to 1:6, about 1:1 to 1:4, or about 1:1 to 1:2.

In one specific example, the antibody-drug conjugate may be a linker-drug conjugate represented by the following chemical formula V, in which a maleimidyl group is conjugated to an antibody.

<Chemical Formula V>

In the above chemical formula V,

L ¹ , L ² , L ³ and L ⁴ may each independently be a direct bond or C _1-10 alkylene.

R ^xa , R ^xb , R ^xc , R ^xd , R ^xe , R ^ya and R ^za can each independently be H or C _1-10 alkyl.

R ¹ may be C _1-10 alkylene.

Z ¹ may be a heteroatom selected from NR ³ , O, S and Se.

R ³ can be H or C _1-8 alkyl.

R ² can be H or oxo (=O).

a and b can each independently be 0 or 1.

c, d and e can each independently be an integer from 0 to 10.

In some embodiments, L ¹ , L ² , L ³ and L ⁴ can each independently be a direct bond; or a straight-chain or branched C _1-10 alkylene. Specifically, L ¹ , L ² , L ³ and L ⁴ can each independently be a C _1-8 alkylene, a C _1-6 alkylene, a C _1-4 alkylene or a C _1-3 alkylene. In some embodiments, L ¹ , L ² , L ³ and L ⁴ can each independently be a straight-chain C _1-6 alkylene, a C _1-4 alkylene or a C _1-3 alkylene. For example, L ¹ , L ² , L ³ and L ⁴ can each independently be methylene or ethylene. For example, L ¹ , L ² and L ³ can be ethylene. For example, L ⁴ could be methylene.

In some embodiments, R ^xa , R ^xb , R ^xc , R ^xd , R ^xe , R ^ya and R ^za can each independently be H or C _1-10 alkyl. Specifically, R ^xa , R ^xb , R ^xc , R ^xd , R ^xe , R ^ya and R ^za can each independently be H, C _1-8 alkyl, C _1-6 alkyl, C _1-4 alkyl or C _1-3 alkyl. For example, R ^xa , R ^xb , R ^xc , R ^xd , R ^xe , R ^ya and R ^za can each independently be H or methyl.

In some embodiments, R ^ya can be C _1-10 alkyl. In some embodiments, R ^ya can be C _1-8 alkyl, C _1-6 alkyl, or C _1-4 alkyl. For example, R ^ya can be methyl.

In some embodiments, R ^ya and R ^za can be the same or different. For example, R ^ya and R ^za can each be methyl. Alternatively, R ^ya can be methyl and R ^za can be H.

In some embodiments, R ¹ can be a straight-chain or branched C _1-10 alkylene. Specifically, R ¹ can be a C _1-8 alkylene, a C _1-6 alkylene, a C _1-4 alkylene, or a C _1-3 alkylene. For example, R ¹ can be propylene or butylene.

In some embodiments, Z ¹ can be a heteroatom selected from NR ³ , O, S, and Se. Specifically, Z ¹ can be O, S, or Se. For example, Z ¹ can be Se.

In some embodiments, R ³ can be H or C _1-8 alkyl. Specifically, R ³ can be H, C _1-6 alkyl, C _1-4 alkyl, or C _1-3 alkyl.

In some embodiments, a and b can each independently be 0 or 1. When a and b are 0, the residues within the parentheses can be absent.

In some embodiments, c, d, and e can each independently be an integer from 0 to 10. When c, d, and e are 0, the residues within the parentheses can be absent. Specifically, c, d, and e can each independently be an integer from 0 to 8, 0 to 6, or 0 to 4. For example, c can be 0 or 2. For example, d can be 0 or 2. For example, e can be 3 or 4.

In one specific example, at least one of c and d may not be 0.

In one specific example, when b is 1, e may not be 0.

In one specific example, the antibody-drug conjugate of the present invention may be represented by the following chemical formula VA.

<Chemical formula VA>

In the above chemical formula VA,

The mAb may be an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.

L ¹ , L ² , L ³ , L ⁴ , R ^xa , R ^xb , R ^xc , R ^xd , R ^xe , R ^ya , R ^za , R ¹ , R ² , Z ¹ , a, b, c, d and e are as described in the above chemical formula V.

In the above chemical formula VA, n is a real number from 1 to 10. Specifically, n may be an integer from 1 to 10.

In one specific example, the antibody-drug conjugate may be a linker-drug conjugate in which a linker and an anticancer agent are conjugated in a 1:1 ratio, and is conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. More specifically, the linker-drug conjugate may be any one selected from the following <Chemical Formula VI>.

<Chemical Formula VI>

In the above chemical formula VI,

The above mAb is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof,

The above m5, m6, m9, m10, m11, m12, n9, n10, n13, n14 and n15 are each independently integers from 1 to 10,

wherein R ^d3 , R ^d5 and R ^d6 are each independently H or C _1-8 alkyl,

wherein Z ¹ is a heteroatom selected from NR ³ , O, S and Se, R ³ is H or C _1-8 hydrocarbyl,

The above D is an anticancer agent,

The above n is a real number between 1 and 10.

In one specific example, m5, m6, m9, m10, m11, m12, n9, n10, n13, n14 and n15 can each independently be an integer from 1 to 10. Specifically, m5, m6, m9, m10, m11, m12, n9, n10, n13, n14 and n15 can each independently be an integer from 1 to 8, 1 to 6, 1 to 4, 1 to 3 or 1 to 2. For example, m5, m6, m9, m10, m11, m12, n9, n10, n13, n14 and n15 can each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In one specific example, R ^d3 , R ^d5 , and R ^d6 can each independently be H or C _1-8 alkyl. Specifically, R ^d3 , R ^d5 , and R ^d6 can each independently be H, C _1-6 alkyl, C _1-4 alkyl, or C _1-3 alkyl. For example, R ^d3 , R ^d5 , and R ^d6 can each independently be H or methyl.

In one specific example, Z ¹ can be a heteroatom selected from NR ³ , O, S, and Se. Specifically, Z ¹ can be O, S, or Se. For example, Z ¹ can be Se.

In one specific embodiment, R ³ can be H or C _1-8 hydrocarbyl. Specifically, R ³ can be H, C _1-6 alkyl, C _1-4 alkyl, or C _1-3 alkyl.

In the above chemical formula VI, n may be a real number from 1 to 10. Specifically, n may be a real number from 1 to 8, a real number from 1 to 6, a real number from 1 to 4, or a real number from 1 to 2. More specifically, n may be an integer from 1 to 10. For example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one specific example, the antibody-drug conjugate may be a linker-drug conjugate in which a linker and an anticancer agent are conjugated in a 1:2 ratio, and is conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. More specifically, the linker-drug conjugate may be any one selected from the following <Chemical Formula VII>.

<Chemical Formula VII>

In the above chemical formula VII,

The above mAb is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof,

The above D is an anticancer drug, and the anticancer drugs may be the same or different.

wherein Z ¹ is a heteroatom selected from NR ³ , O, S and Se, R ³ is H or C _1-8 hydrocarbyl,

The above q1 to q3 are each independently an integer from 0 to 10, and the above q4 is an integer from 1 to 10,

wherein R ^d1 , R ^d5 and R ^e are each independently H or C _1-8 alkyl,

The above n1, n3, n4, n8, n14 and n15, m2, m8 and m10 are each independently integers from 1 to 8,

The above n is a real number between 1 and 10.

In one specific example, Z ¹ can be a heteroatom selected from NR ³ , O, S, and Se. Specifically, Z ¹ can be O, S, or Se. For example, Z ¹ can be Se.

In one specific embodiment, R ³ can be H or C _1-8 hydrocarbyl. Specifically, R ³ can be H, C _1-6 alkyl, C _1-4 alkyl, or C _1-3 alkyl.

In one specific example, q1 to q3 can each independently be an integer from 0 to 10. Specifically, q1 to q3 can each independently be an integer from 1 to 8, an integer from 1 to 6, an integer from 1 to 4, or an integer from 1 to 2. For example, q1 to q3 can each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one specific example, q4 can be an integer from 1 to 10. Specifically, q4 can be an integer from 1 to 8, an integer from 1 to 6, an integer from 1 to 4, or an integer from 1 to 2. For example, q4 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one specific example, n1, n3, n4, n8, n14 and n15, m2, m8 and m10 are each independently integers from 1 to 8, and specifically, n1, n3, n4, n8, n14 and n15, m2, m8 and m10 can each independently be an integer from 1 to 8, an integer from 1 to 6, an integer from 1 to 4 or an integer from 1 to 2. For example, n1, n3, n4, n8, n14 and n15, m2, m8 and m10 can each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In the above chemical formula VII, n may be a real number from 1 to 10. Specifically, n may be a real number from 1 to 8, a real number from 1 to 6, a real number from 1 to 4, or a real number from 1 to 2. More specifically, n may be an integer from 1 to 10. For example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

At this time, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is the same as described above. In addition, the anticancer agents may be the same or different.

As a specific example, TAB07.409.1 ADC, an antibody-drug conjugate comprising LT409 conjugated to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, may have the following structure:

.

At this time, n can be a real number from 1 to 10. Specifically, n can be an integer from 1 to 10.

Pharmaceutical composition comprising an antibody-drug conjugate

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-CLDN18.2 antibody or antigen-binding fragment thereof-drug conjugate as an active ingredient.

Another aspect of the present invention provides a use of an anti-CLDN18.2 antibody or antigen-binding fragment thereof-drug conjugate for the prevention or treatment of cancer.

Another aspect of the present invention provides a method for preventing or treating cancer comprising administering to a subject an anti-CLDN18.2 antibody or antigen-binding fragment thereof-drug conjugate.

At this time, the anti-CLDN18.2 antibody or antigen-binding fragment thereof-drug conjugate, cancer, prevention, treatment, subject and administration are the same as described above.

Hereinafter, the present invention will be specifically described by way of examples. However, these examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not construed as being limited by these examples.

The meanings of the abbreviations used are as follows:

MC: Methylene chloride

ACN: Acetonitrile

EA: Ethyl Acetate

THF: Tetrahydrofuran

DMF: Dimethylformamide

TBAI: Tetrabutylammonium iodide

HMTETA: 1,1,4,7,10,10-hexamethyltriethylenetetramine

DIPEA: Diisopropylethylamine

HOBt: 1-hydroxybenzotriazole

HATU: Hexafluorophosphate azabenzotriazole tetramethyl uronium

EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

DCC: N,N'-dicyclohexylcarbodiimide

LAH: lithium aluminum borohydride

Manufacturing Example 1: Preparation of Compound A-6

Step 1: Preparation of Compound A-2

Compound A-1 (Fmoc-sar-sar-sar-OH, CAS No. 2749824-37-9, 500 mg, 1.10 mmol) was dissolved in tert-butanol (10 mL) at room temperature under a nitrogen atmosphere. Di-tert-butyl dicarbonate (722 mg, 3.31 mmol) and 4-dimethylaminopyridine (53.8 mg, 0.44 mmol) were added, and the mixture was stirred at room temperature for 16 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the organic layer was extracted twice with EA (20 mL), distilled water (10 mL), and aqueous sodium chloride solution (10 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound A-2 as a white solid (516 mg, 92%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.71 - 7.73 (m, 2H), 7.63 - 7.61 (d, J = 7.2 Hz, 2H), 7.41 - 7.37 (m, 2H), 7.32 - 7.29 (m, 2H), 4.38 - 4.37 (m, 1H), 4.29 (s, 2H), 4.24 (s, 2H), 4.09 (s, 2H), 3.99 - 3.93 (m, 2H), 3.08 (s, 3H), 3.04 (s, 3H), 2.94 (s, 3H), 1.45 (s, 9H); EI-MS m/z: 510 [M+H] ⁺ .

Step 2: Preparation of Compound A-3

Compound A-2 (747 mg, 1.46 mmol) was dissolved in MC (10 mL) at 0°C under a nitrogen atmosphere, diethylamine (2 mL, 20% v/v) was slowly added dropwise, and the reaction solution was stirred for 30 minutes. Additionally, the mixture was stirred at room temperature for 3 hours, and the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound A-3 as a yellow oil (388 mg, 92%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.27 (s, 2H), 4.14 (bs, 1H), 4.01 (s, 2H), 3.47 (s, 2H), 3.06 (s, 3H), 3.04 (s, 3H), 2.45 (s, 3H), 1.46 (s, 9H); EI-MS m/z: 288 [M+H] ⁺ .

Step 3: Preparation of Compound A-4

Compound A-3 (385 mg, 1.34 mmol) was dissolved in MeOH (20 mL) at 0°C under a nitrogen atmosphere, and 37% formaldehyde solution (0.6 mL, 8.04 mmol) and acetic acid (1.53 mL, 26.82 mmol) were added. The mixture was stirred for 20 minutes, and then sodium cyanoborohydride (337.1 mg, 5.36 mmol) was added and reacted at room temperature for 16 hours. After completion of the reaction, the residue obtained by concentration under reduced pressure was purified by column chromatography to obtain compound A-4 as a transparent oil (337.6 mg, 83%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.27 (s, 2H), 4.01 (s, 2H), 3.36 (s, 2H), 3.10 (s, 3H), 3.05 (s, 3H), 2.43 - 2.42 (m, 6H), 1.45 (s, 9H); EI-MS m/z: 302 [M+H] ⁺ .

Step 4: Preparation of Compound A-5

MC (10 mL) was dissolved in compound A-4 (1.0 g, 3.32 mmol) at 0°C under a nitrogen atmosphere, and trifluoroacetic acid (2 mL, 20% v/v) was slowly added dropwise. The reaction solution was stirred at room temperature for 16 h. After the reaction, the solution was concentrated under reduced pressure, and the residue was diluted with distilled water (50 mL) and ACN (20 mL) and lyophilized to obtain compound A-5 as a yellow oil (1.12 g, 94%).

¹ H-NMR (400 MHz, D ₂ O) δ 4.35 (s, 2H), 4.26 (s, 2H), 4.10 (s, 2H), 3.05 (s, 3H), 2.95 (s, 3H), 2.90 (s, 6H); EI-MS m/z: 246 [M+H] ⁺ .

Step 5: Preparation of Compound A-6

Compound A-5 (83.7 mg, 0.233 mmol) was dissolved in DMF (1 mL) at 0°C under a nitrogen atmosphere, and bis(pentafluorophenyl)carbonate (CAS No. 59483-84-0, 110.2 mg, 0.28 mmol) and DIPEA (72 μL, 0.412 mmol) were sequentially added, and the mixture was stirred at 0°C for 30 min. After completion of the reaction, the mixture of compound A-6 was used in the next reaction without further purification. EI-MS m/z: 412 [M+H] ⁺ .

Manufacturing Example 2: Preparation of Compound B-18

Step 1: Preparation of Compound B-2

Compound B-1 (2-chloro-6-hydroxybenzaldehyde, CAS No. 18362-30-6, 500 mg, 3.19 mmol) was dissolved in ACN (10 mL) at 0°C under a nitrogen atmosphere, and benzyl bromide (400 μL, 3.35 mmol) and K ₂ CO ₃ (1.1 g, 7.98 mmol) were added, and the mixture was stirred at room temperature for 16 h. After completion of the reaction, EA (100 mL) and distilled water (100 mL) were added to extract the organic layer, and the obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-2 as a white solid (750 mg, 95%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 10.57 (s, 1H), 7.45 - 7.34 (m, 6H), 7.04 (d, J = 8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 5.19 (s, 2H).

Step 2: Preparation of Compound B-3

Selenium powder (256 mg, 3.24 mmol) was added to THF (10 mL) at room temperature under a nitrogen atmosphere, cooled to 0°C, and n-butyllithium solution (2.5 M in hexane, 1.42 mL, 3.56 mmol) was slowly added dropwise. The mixture was stirred at 0°C for 40 min, then compound B-2 (800 mg, 3.24 mmol) dissolved in DMF (2 mL) was added, and stirred at room temperature for 12 h. After completion of the reaction, the mixture was cooled to 0°C, and distilled water (100 mL) was slowly added dropwise to terminate the reaction. After the reaction, EA (100 mL) was added to the solution, and the organic layer was extracted, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound B-3 (900 mg, 80%) as a light yellow solid.

^1H -NMR (400 MHz, CDCl ₃ ) δ 10.70 (s, 1H), 7.42 - 7.33 (m, 6H), 7.04 (d, J = 8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 5.18 (s, 2H), 2.85 (t, J = 7.6 Hz, 2H), 1.75 (q, J = 7.6 Hz, 2H), 1.51 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H).

Step 3: Preparation of Compound B-4

Compound B-3 (900 mg, 2.59 mmol) was dissolved in DMF (10 mL) at room temperature under a nitrogen atmosphere, ethyl bromoacetate (574 μL, 6.48 mmol) was added, and the mixture was stirred at 120°C for 12 h. After the reaction, the solution was cooled to room temperature and used in the next reaction without further purification.

Step 4: Preparation of Compound B-5

After the reaction of step 3 containing compound B-4 under a nitrogen atmosphere, K ₂ CO ₃ (716 mg, 6.48 mmol) was added to the solution and stirred at 120°C for 2.5 hours. After completion of the reaction, EA (200 mL) and distilled water (200 mL) were added to extract the organic layer. The obtained organic layer was washed again with distilled water (200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-5 as a white solid (840 mg, 90%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.55 (s, 1H), 7.49 - 7.47 (m, 3H), 7.43 - 7.29 (m, 4H), 6.84 (d, J = 8 Hz, 1H), 5.21 (s, 2H), 4.37 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H); EI-MS m/z: 361 [M+H] ⁺ .

Step 5: Preparation of Compound B-6

LAH (2.25 g, 59.29 mmol) was added to THF (300 mL) at room temperature under a nitrogen atmosphere, and the mixture was cooled to 0°C. Compound B-5 (8.53 g, 23.74 mmol) was dissolved in THF (75 mL), slowly added, and stirred at 0°C for 5 minutes. After completion of the reaction, 2 N aqueous sodium hydroxide solution (36 mL) was added to terminate the reaction, and the reaction solution was diluted with THF (150 mL) and filtered using Celite. The obtained solution was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound B-6 as an ivory-colored solid (7.2 g, 96%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.66 (s, 1H), 7.48 - 7.33 (m, 6H), 7.19 (t, J = 8 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 5.19 (s, 2H), 4.93 (d, J = 6) Hz, 2H), 1.92 (t, J = 6 Hz, 1H); EI-MS m/z: 318 [M+H] ⁺ .

Step 6: Preparation of Compound B-7

Compound B-6 (4.66 g, 14.69 mmol) was dissolved in MC (93 mL) at room temperature under a nitrogen atmosphere, and then TEMPO (CAS No. 2564-83-2, 230 mg, 1.47 mmol) and TBAI (CAS No. 311-28-4, 543 mg, 1.47 mmol) were added. An aqueous solution of NaHCO ₃ (4.2 g, 0.5 mol) and K ₂ CO ₃ (691 mg, 0.05 mol) dissolved in distilled water (100 mL) and N -chlorosuccinimide (CAS No. 128-09-6, 2.16 g, 16.18 mmol) were sequentially added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, MC (100 mL) and distilled water (100 mL) were added to extract the organic layer twice, and the organic layer was washed with aqueous sodium chloride solution (100 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-7 as a yellow solid (4.35 g, 94%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 9.95 (s, 1H), 8.53 (s, 1H), 7.53 - 7.35 (m, 7H), 6.88 (d, J = 8 Hz, 1H), 5.23 (s, 2H); EI-MS m/z: 316 [M+H] ⁺ .

Step 7: Preparation of Compound B-8

Compound B-7 (8 g, 25.38 mmol) was dissolved in MC (242 mL, 0.105 M) at -78°C under a nitrogen atmosphere, and then BCl ₃ solution (1.0 M in MC, 76.14 mL, 76.14 mmol) was slowly added and stirred for 2 h. After completion of the reaction, distilled water (200 mL) and 2 N sodium hydroxide aqueous solution were slowly added dropwise at 0°C to adjust the pH to 10, and then MC (50 mL) was added to wash the aqueous layer. 2 N hydrochloric acid aqueous solution was slowly added to the obtained aqueous layer to adjust the pH of the reaction solution to 3 to 4, and then the organic layer was extracted twice using EA (200 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain compound B-8 as a light green solid (4.9 g, 85.8%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 9.97 (s, 1H), 8.65 (s, 1H), 7.53 (d, J = 8 Hz, 1H), 7.32 (t, J = 8 Hz, 1H), 6.82 (d, J = 8 Hz, 1H); EI-MS m/z: 226 [M+H] ⁺ .

Step 8: Preparation of Compound B-9

Silver carbonate (93.8 g, 340 mmol) and HMTETA (13.7 mL, 50.5 mmol) were dissolved in ACN (700 mL) at room temperature under a nitrogen atmosphere and stirred for 1 hour. After the reaction solution was cooled to 0°C, a solution of compound B-8 (20.7 g, 91.9 mmol) in THF (300 mL) and acetobromo-α-D-glucuronic acid methyl ester (CAS No. 21085-72-3, 8.82 g, 22.2 mmol) were sequentially added slowly, and the mixture was stirred for 16 hours while gradually increasing the temperature from 0°C to room temperature. After completion of the reaction, the mixture was filtered using Celite, the filtrate was concentrated under reduced pressure, and the organic layer was extracted twice by adding EA (1,000 mL) and distilled water (1,000 mL). The obtained organic layer was washed with an aqueous sodium chloride solution (1,000 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain compound B-9 as a yellow solid (35 g, 70.4%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 9.97 (s, 1H), 8.36 (s, 1H), 7.64 (d, J = 8 Hz, 1H), 7.39 (t, J = 8 Hz, 1H), 6.97 (d, J = 8 Hz, 1H), 5.5 - 5.36 (m, 3H), 5.31 (m, 1H), 4.25 (m, 1H), 3.73 (s, 3H), 2.09 (s, 3H), 2.07 (s, 6H); EI-MS m/z: 542 [M+H] ⁺ .

Step 9: Preparation of Compound B-10

Compound B-9 (35 g, 64.6 mmol) was dissolved in THF (650 mL) at 0°C under a nitrogen atmosphere, and sodium borohydride (3.67 g, 96.9 mmol) was added and stirred for 2 hours. After completion of the reaction, distilled water (500 mL) was added to terminate the reaction, and EA (500 mL) was added to extract the organic layer twice. The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-10 as a white solid (26 g, 74%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.58 (d, J = 8 Hz, 1H), 7.47 (s, 1H), 7.2 (t, J = 8 Hz, 1H), 6.94 (d, J = 8 Hz, 1H), 5.41 - 5.35 (m, 3H), 5.23 (m, 1H), 4.93 (d, J = 6 Hz, 2H), 4.21 (m, 1H), 3.73 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.99 (t, J = 6 Hz, 1H); EI-MS m/z: 566 [M+Na] ⁺ .

Step 10: Preparation of Compound B-11

Compound B-10 (700 mg, 1.29 mmol) was dissolved in concentrated HCl (35 mL) under a nitrogen atmosphere at -10°C and stirred for 2 h at -10°C. After completion of the reaction, EA (40 mL) and distilled water (40 mL) were added, and the organic layer was extracted twice. The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound B-11 as a white solid (700 mg, 97.2%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.6 - 7.54 (m, 2H), 7.23 - 7.2 (m, 1H), 6.95 (d, J = 8 Hz, 1H), 5.5 - 5.35 (m, 3H), 5.22 (d, J = 7.2 Hz, 1H), 4.88 (s, 2H), 4.23 - 4.2 (m, 1H), 3.73 (s, 3H), 2.08 - 2.04 (m, 9H); EI-MS m/z: 584 [M+Na] ⁺ .

Step 11: Preparation of Compound B-12

Compound B-11 (24.9 g, 44.4 mmol) was dissolved in DMF (880 mL) at room temperature under a nitrogen atmosphere, sodium azide (4.33 g, 66.6 mmol) was added, and the mixture was stirred at 60°C for 17 hours. After completion of the reaction, distilled water (1,000 mL) was added, and the mixture was extracted twice with EA (1,000 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-12 as a white solid (17.3 g, 68.9%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.58 (d, J = 8 Hz, 1H), 7.52 (s, 1H), 7.25 - 7.2 (m, 1H), 6.95 (d, J = 8 Hz, 1H), 5.46 - 5.34 (m, 3H), 5.24 (d, J = 7.2 Hz, 1H), 4.63 (d, J = 14.4 Hz, 1H), 4.57(d, J = 14.4 Hz, 1H) 4.26 - 4.19 (m, 1H), 3.73 (s, 3H), 2.08 (s, 3H), 2.06 (s, 6H); EI-MS m/z: 591 [M+Na] ⁺ .

Step 12: Preparation of Compound B-13

B-12 (17.3 g, 30.2 mmol) was dissolved in MC (950 mL) at -78°C under a nitrogen atmosphere, then dichloromethyl methyl ether (CAS No. 4885-02-3, 27.5 mL, 302 mmol) and titanium (IV) chloride solution (1.0 M in MC, 302 mL, 302 mmol) were sequentially added slowly and the mixture was stirred while maintaining the temperature for 6 h. After completion of the reaction, EA (1,500 mL) was added at -78°C to dilute the reaction solution, then cooled distilled water (1,000 mL) was slowly added, and the organic layer was extracted twice. The obtained organic layer was washed with aqueous sodium chloride solution (1,000 mL). The washed organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain compound B-13 as a white solid (8.5 g, 46.4%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 10.13 (s, 1H), 7.82 (d, J = 8 Hz, 1H), 7.6 (s, 1H), 7.12 (d, J = 8 Hz, 1H), 5.5 - 5.38 (m, 4H), 4.7 (d, J = 14.4 Hz, 1H), 4.63 (d, J = 14.4 Hz, 1H), 4.35 - 4.27 (m, 1H), 3.72 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H); EI-MS m/z: 619 [M+Na] ⁺ .

Step 13: Preparation of Compound B-14

Compound B-13 (9.3 g, 15.6 mmol) was dissolved in THF (623 mL) at 0°C under a nitrogen atmosphere, sodium borohydride (885 mg, 23.4 mmol) was added, and the mixture was stirred for 3 hours. After completion of the reaction, distilled water (500 mL) was added to quench the reaction, EA (1,000 mL) was added, and the organic layer was extracted twice, followed by washing with aqueous sodium chloride solution (1,000 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-14 as a white solid (6.6 g, 71%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.56 (s, 1H), 7.2 (d, J = 8 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 5.45 - 5.3 (m, 3H), 5.22 (d, J = 6.8 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 4.63 (d, J = 14.4 Hz, 1H), 4.58 (d, J = 14.4 Hz, 1H), 4.24 - 4.2 (m, 1H), 3.74 (s, 3H), 2.08 (s, 3H), 2.05 (s, 6H), 1.74 (t, J = 6 Hz, 1H); EI-MS m/z: 621 [M+Na] ⁺

Step 14: Preparation of Compound B-15

Compound B-14 (423 mg, 0.71 mmol) was dissolved in MC (18.5 mL) at 0°C under a nitrogen atmosphere, and 4-nitrophenylchloroformate (285 mg, 1.41 mmol), pyridine (170 μL, 2.12 mmol), and DIPEA (180 μL, 1.06 mmol) were sequentially added. The mixture was stirred at 0°C for 30 minutes and at room temperature for 2 hours. After completion of the reaction, the mixture was diluted with MC (50 mL), and 2 N aqueous hydrochloric acid solution (50 mL) was added. The organic layer was extracted twice, and then aqueous sodium chloride solution (60 mL) was added to wash the organic layer. The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound B-15 as a white solid (499.2 mg, 92.6%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.27 (d, J = 9.2 Hz, 2H), 7.58 (s, 1H), 7.38 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 8 Hz, 1H), 6.98 (d, J = 8 Hz, 1H), 5.46 - 5.34 (m, 5H), 5.27 (d, J = 7.2 Hz, 1H), 4.66 (d, J = 14.4 Hz, 1H), 4.6 (d, J = 14.4 Hz, 1H), 4.26 - 4.22 (m, 1H), 3.74 (s, 3H), 2.08 (s, 3H), 2.07 2.03 (m, 6H); EI-MS m/z: 786 [M+Na] ⁺ .

Step 15: Preparation of Compound B-16

Compound B-15 (53.7 mg, 0.07 mmol) and Exatecan mesylate (CAS No. 169869-90-3, 37.4 mg, 0.07 mmol) were dissolved in DMF (2 mL) at 0°C under a nitrogen atmosphere. HOBt (10.4 mg, 0.08 mmol), pyridine (0.3 mL), and DIPEA (24.5 μL, 0.14 mmol) were sequentially added, and the mixture was stirred at 0°C for 30 min. Additionally, the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, the organic layer was extracted using EA (50 mL) and 2 N hydrochloric acid aqueous solution (50 mL), and then washed with sodium chloride aqueous solution (50 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain compound B-16 as a yellow solid (66.8 mg, 89%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.67 (m, 1H), 7.56 (s, 1H), 7.48 (s, 1H), 7.26 (m, 1H), 6.84 (d, J = 8 Hz, 1H), 5.68 (m, 1H), 5.45 - 5.23 (m, 9H), 5.11 (m, 1H), 4.57 (s, 2H), 4.15 (m, 1H), 3.73 (s, 3H), 3.20 - 3.13 (m, 2H), 2.42 (s, 3H), 2.39 - 2.28 (m, 2H), 2.11 - 2.04 (m, 10H), 1.90 (q, J = 7.2 Hz, 2H), 1.28 - 1.23 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H); EI-MS m/z: 1060 [M+H] ⁺ .

Step 16: Preparation of Compound B-17

Under a nitrogen atmosphere at -20°C, compound B-16 (66.8 mg, 0.063 mmol) was dissolved by adding a mixture of MeOH (3.2 mL) and THF (3.2 mL). Lithium hydroxide monohydrate (13.2 mg, 0.315 mmol) dissolved in distilled water (0.64 mL) was added dropwise, and the mixture was stirred for 4 hours while gradually raising the temperature from -20°C to -5°C. After completion of the reaction, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 2 to 3, and the mixture was diluted with ACN (1 mL) and distilled water (3 mL). The mixture was separated, purified, and lyophilized using Preparative-HPLC to obtain compound B-17 as an ivory solid (36.3 mg, 63%). EI-MS m/z: 920 [M+H] ⁺ .

Step 17: Preparation of Compound B-18

Compound B-17 (20.9 mg, 0.023 mmol) was dissolved in 1,4-dioxane (2 mL) and distilled water (0.2 mL) at room temperature under a nitrogen atmosphere. Tributylphosphine (13.8 mg, 0.068 mmol) was added and stirred for 2 h. After completion of the reaction, the mixture was diluted with ACN (1 mL) and distilled water (3 mL), and the residue was separated, purified, and lyophilized using Preparative-HPLC to obtain compound B-18 (11.5 mg, 56%). EI-MS m/z: 894 [M+H]+.

Manufacturing Example 3: Preparation of Compound D-3

Step 1: Preparation of compound D-2

Compound D-1 (Mal-PEG2-acid, CAS No. 1374666-32-6, 500 mg, 1.94 mmol) was dissolved in MC (15 mL) at 0°C under a nitrogen atmosphere, N-hydroxysuccinimide (246 mg, 2.14 mmol) and DCC (441 mg, 2.14 mmol) were added, and the mixture was stirred for 15 hours. After completion of the reaction, EA (10 mL) and hexane (10 mL) were added, and the resulting precipitate was removed by filtration. The filtrate was concentrated, and EA (10 mL) and hexane (10 mL) were added again to remove the resulting precipitate, and the filtrate was concentrated under reduced pressure to obtain compound D-2 (689 mg, 100%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 6.70 (s, 2H), 3.81 (t, J = 6.4 Hz, 2H), 3.72 (m, 2H), 3.65 - 3.58 (m, 6H), 2.87 (t, J = 6.4 Hz, 2H), 2.84 (s) 4H); EI-MS m/z: 355 [M+H] ⁺ .

Step 2: Preparation of compound D-3

Lys(Boc)-OH (CAS No. 2418-95-3, 206 mg, 0.837 mmol) was stirred in DMF (5 mL) at 0°C for 10 minutes under a nitrogen atmosphere at room temperature. Compound D-2 (356 mg, 1.01 mmol) and DIPEA (291 μL, 1.67 mmol) were slowly added to the cooled white suspension, and the mixture was stirred at 0°C for 30 minutes and at room temperature for 3 hours. After completion of the reaction, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 4 to 5, and the reaction solution was diluted with distilled water (5 mL). The residue was separated, purified, and lyophilized using Preparative-HPLC to obtain compound D-3 (353 mg, 87%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.11 (d, J = 7.2 Hz, 1H), 6.72 (s, 2H), 4.74 (brs, 1H), 4..54 (bs, 1H), 3.76 - 3.71 (m, 4H), 3.65 - 3.60 (m, 6H), 3.12 - 3.10 (m, 2H), 2.52 (t, J = 5.2 Hz, 2H), 1.91 (bs, 1H), 1.76 (bs, 1H), 1.50 (m, 2H), 1.44 (s, 9H), 1.39 (m, 2H); EI-MS m/z: 486 [M+H] ⁺ .

Manufacturing Example 4: Preparation of Compound E-4

Step 1: Preparation of Compound E-2

Compound E-1 (Fmoc-sar-sar-sar-OH, CAS No. 2749824-37-9, 300 mg, 0.66 mmol) was dissolved in MC (9.5 mL) at 0°C under a nitrogen atmosphere, piperidine (0.5 mL) was added, and the mixture was stirred at 0°C for 10 minutes. The mixture was slowly warmed to room temperature and stirred for 2.5 hours. After the reaction, distilled water (10 mL) was added to the solution, washed three times with ether (30 mL), and the aqueous layer was concentrated under reduced pressure to obtain compound E-2 as a white solid (253 mg, 100%).

¹ H-NMR (400 MHz, D ₂ O) δ 4.37 (s, 1H), 4.17 (m, 1H), 4.07 (m, 1H), 3.88 (m, 2H), 2.98 (m, 3H), 2.94 (s, 1H), 2.90 (m, 2H), 2.71 (s, 2H), 2.67 (s, 1H); EI-MS m/z: 232 [M+H] ⁺ .

Step 2: Preparation of compound E-3

Compound E-2 (253 mg, 1.09 mmol) was dissolved in distilled water (2 mL) at room temperature under a nitrogen atmosphere, and NaHCO ₃ (276 mg, 3.29 mmol) and di-tert-butyl dicarbonate (1.2 g, 5.50 mmol) were added, followed by stirring at room temperature for 16 h. After completion of the reaction, the mixture was diluted with distilled water (6 mL), and then separated, purified, and lyophilized using Preparative-HPLC to obtain compound E-3 as a white solid (188 mg, 85.8%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.27 (s, 2H), 4.13 (m, 2H), 4.09 - 4.06 (m, 2H), 3.07 (m, 4H), 3.00 - 2.97 (m, 3H), 2.93 - 2.90 (m, 3H), 1.47 - 1.43 (m, 9H); EI-MS m/z: 332 [M+H] ⁺ . ^.

Step 3: Preparation of compound E-4

Compound E-3 (10 mg, 0.030 mmol) was dissolved in MC (1 mL) at 0°C under a nitrogen atmosphere, and EDC (6 mg, 0.031 mmol) and N-hydroxysuccinimide (4 mg, 0.035 mmol) were added, followed by stirring for 5 h. After completion of the reaction, the mixture of compound E-4 was used in the next reaction without further purification. EI-MS m/z: 429 [M+H] ⁺ .

Synthesis Example 1: Preparation of LT409

Step 1: Preparation of compound C-2

Under a nitrogen atmosphere, DMF (7 mL) was added to Lys(Boc)-OH (CAS No. 2418-95-3, 1.27 g, 5.17 mmol) at room temperature and stirred at 0°C for 10 min. To the cooled white suspension, compound C-1 (maleimide-PEG2-NHS ester, CAS No. 955094-26-5, 2.0 g, 4.70 mmol) and DIPEA (1.64 mL, 9.4 mmol) were slowly added and stirred at 0°C for 2 h and at room temperature for 3 h. After completion of the reaction, the solution was diluted with distilled water (30 mL) at 0°C and 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 2 to 3. Distilled water (170 mL) was added to the reaction solution at room temperature and extracted five times with EA (200 mL). The obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound C-2 in the form of a sticky pale yellow oil (2.27 g, 57.91%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.14 (d, J = 7.2 Hz, 1H), 6.71 (s, 2H), 4.72 (brs, 1H), 4.62 (brs, 1H), 3.92 - 3.89 (m, 2H), 3.89 - 3.78 (m, 2H), 3.62 (s, 3H), 3.60 - 3.43 (m, 3H), 3.39 - 3.30 (m, 1H), 3.17 - 3.04 (m, 2H), 2.63 - 2.53 (m, 4H), 1.91 - 1.88 (m, 1H), 1.79 - 1.74 (m, 1H), 1.51 - 1.48 (m, 2H), 1.44 (s, 9H); EI-MS m/z: 557 [M+H] ⁺ .

Step 2: Preparation of compound C-3

Compound C-2 (455.5 mg, 0.818 mmol) was dissolved in DMF (4 mL) at 0°C under a nitrogen atmosphere, stirred for 10 minutes, and HATU (339.45 mg, 0.893 mmol), amino-PEG2-acid tert-butyl ester (CAS No. 756525-95-8, 166.89 μL, 0.744 mmol), and DIPEA (0.26 mL, 1.49 mmol) were sequentially added, and stirred at 0°C for 1 hour. After completion of the reaction, the mixture was slowly added dropwise to a 2 N hydrochloric acid aqueous solution at 0°C to adjust the pH of the reaction solution to 4 to 5, diluted with distilled water (6 mL) and ACN (4 mL), and separated, purified, and lyophilized using Preparative-HPLC to obtain Compound C-3 as a white solid (297.6 mg, 51.82%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.25 - 7.20 (brs, 1H), 7.04 (d, J = 7.2 Hz, 1H), 6.92 - 6.86 (m, 1H), 6.70 (s, 2H), 4.84 - 4.75 (brs, 1H), 4.50 - 4.41 (m, 1H), 3.85 (t, J = 7.2 Hz, 2H), 3.78 - 3.68 (m, 4H), 3.67 - 3.57 (m, 8H), 3.57 - 3.50 (m, 4H), 3.50 - 3.39 (m, 3H), 3.3 - 3.29 (m, 1H), 3.15 - 3.03 (m, 2H), 2.57 (t, J = 7.2 Hz, 2H), 2.54 - 2.46 (m, 4H), 1.89 - 1.75 (m, 2H), 1.55 - 1.39 (m, 20H), 1.39 - 1.31 (m, 2H); EI-MS m/z: 772 [M+H] ⁺ .

Step 3: Preparation of compound C-4

Compound C-3 (194.0 mg, 0.25 mmol) was dissolved in MC (3.5 mL) at 0°C under a nitrogen atmosphere and cooled for 10 min. Trifluoroacetic acid (1.5 mL) was slowly added dropwise to the reaction solution and stirred at 0°C for 7 h. After completion of the reaction, the mixture was diluted with MC (100 mL) and concentrated under reduced pressure three times below 15°C. The concentrated residue was diluted with ACN (100 mL), further concentrated twice below 25°C, diluted with toluene (100 mL), concentrated once below 25°C, and then concentrated under reduced pressure for 1 h. After diluting with distilled water (50 mL) and ACN (20 mL), the mixture was lyophilized to obtain the TFA salt of compound C-4 as a clear oil (197.5 mg, 107.6%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.90 - 7.73 (brs, 2H), 7.40 - 7.30 (m, 2H), 7.22 - 7.16 (m, 1H), 6.71 (s, 2H), 4.70 - 4.62 (brs, 1H), 3.85 - 3.72 (m, 5H), 3.72 - 3.65 (m, 1H), 3.65 - 3.58 (m, 8H), 3.58 - 3.50 (m, 4H), 3.50 - 3.32 (m, 4H), 3.15 - 2.96 (m, 2H), 2.65 - 2.58 (m, 2H), 2.58 - 2.49 (m, 4H), 2.0 - 1.85 (m, 2H), 1.60 - 1.40 (m, 4H); EI-MS m/z: 616 [M+H] ⁺ .

Step 4: Preparation of compound C-5

Compound C-4 (170 mg, 0.233 mmol) was dissolved in DMF (1 mL) at 0°C under a nitrogen atmosphere, and then the mixture of compound A-6 and DIPEA (81 μL, 0.464 mmol) were sequentially added, and the mixture was stirred at 0°C for 2 h. After completion of the reaction, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 4 to 5, and the reaction solution was diluted with distilled water (7 mL). The residue was separated, purified, and lyophilized using Preparative-HPLC to obtain compound C-5 as a white solid (130.3 mg, 66.3%). EI-MS m/z: 843 [M+H] ⁺ , 422 1/2 [M+H] ⁺ .

Step 5: Fabrication of the LT409

Compound C-5 (21.8 mg, 0.0259 mmol) was dissolved in DMF (0.5 mL) under a nitrogen atmosphere, and bis(pentafluorophenyl)carbonate (CAS No. 59483-84-0, 12.2 mg, 0.0310 mmol) and N-methylmorpholine (6 μL, 0.0514 mmol) were sequentially added at 0°C, and the mixture was stirred at 0°C for 30 min. After the reaction was completed, compound B-18 (NH ₂ -SIG-Exatecan, 21.0 mg, 0.0235 mmol), N-methylmorpholine (5 μL, 0.047 mmol), and distilled water (10 μL) were sequentially added to THF (0.5 mL) and 1,4-dioxane (0.5 mL) without any additional purification process, and the mixture was stirred at 0°C for 30 minutes and then at room temperature for 2 hours. After the reaction was completed, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 4 to 5, and the reaction solution was diluted with distilled water (7 mL), followed by separation, purification, and lyophilization using Preparative-HPLC to obtain LT409 as a white solid (21.8 mg, 54.0%). EI-MS m/z: 1719 [M+H] ⁺ , 860 1/2 [M+H] ⁺ , 573 1/3 [M+H] ⁺ .

Synthesis Example 2: Preparation of LT398

Step 1: Preparation of compound D-4

Compound D-3 (227 mg, 0.468 mmol) was dissolved in MC (5 mL) at 0°C under a nitrogen atmosphere and stirred for 10 min. HATU (282 mg, 0.741 mmol), amino-PEG2-acid tert-butyl ester (CAS No. 756525-95-8, 120 mg, 0.514 mmol), and DIPEA (163 μL, 0.935 mmol) were sequentially added to the reaction solution and stirred at 0°C for 2 h. After completion of the reaction, the mixture was adjusted to pH 4 to 5 by slowly adding 2 N hydrochloric acid aqueous solution dropwise at 0°C, diluted with distilled water (4 mL) and ACN (2 mL), and subjected to preparative-HPLC separation, purification, and lyophilization to obtain compound D-4 as a white solid (152 mg, 46%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 6.84 (d, J = 7.2 Hz, 1H), 6.72 (s, 2H), 6.61 (bs, 1H), 4.72 (bs, 1H), 4.44 - 4.39 (m, 1H), 3.74 - 3.70 (m, 6H), 3.66 - 3.65 (m, 2H), 3.63 - 3.59 (m, H), 3.55 - 3.52 (m, 2H), 3.43 (m, 2H), 3.09 - 3.08 (m, 2H), 2.53 - 2.46 (m, 4H), 1.91 - 1.83 (m, 1H), 1.66 - 1.61 (m, 1H), 1.52 - 1.48 (m, 2H), 1.45 - 1.43 (m, 18H), 1.40 - 1.34 (m, 2H); EI-MS m/z: 701 [M+H] ⁺

Step 2: Preparation of compound D-5

Compound D-4 (152.0 mg, 0.217 mmol) was dissolved in MC (5 mL) at 0°C under a nitrogen atmosphere and cooled for 10 min. Trifluoroacetic acid (1 mL) was slowly added dropwise to the reaction solution, and the mixture was stirred at 0°C for 30 min and at room temperature for 2 h. After completion of the reaction, the mixture was diluted with MC (40 mL) and concentrated three times below 5°C. The mixture was diluted with ACN (40 mL), concentrated twice below 25°C, diluted with toluene (30 mL), and depressurized below 25°C. After diluting with distilled water (80 mL) and ACN (20 mL), the mixture was lyophilized to obtain the TFA salt of compound D-5 as a clear oil (143 mg, 100%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.48 (d, J = 8 Hz, 1H), 7.37 (s, 1H), 6.73 (s, 2H), 4.57 - 4.56 (m, 1H), 3.74 - 3.65 (m, 6H), 3.62 - 3.56 (m, 11H), 3.43 - 3.41 (m, 2H), 3.04 (bs, 2H), 2.64 - 2.59 (m, 3H), 2.52 - 2.49 (m, 3H), 1.86 - 1.68 (m, 5H), 1.49 - 1.44 (m, 3H); EI-MS m/z: 545 [M+H] ⁺ .

Step 3: Preparation of compound D-6

Compound D-5 (55.7 mg, 0.10 mmol) was dissolved in DMF (1 mL) at 0°C under a nitrogen atmosphere, and then the post-reaction solution of step 3 of Preparation Example 1 containing compound A-4 and DIPEA (36 μL, 0.20 mmol) were sequentially added, and the mixture was stirred at 0°C for 1.5 hours. After completion of the reaction, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 4 to 5, and the mixture was diluted with distilled water (6 mL). The mixture was separated, purified, and lyophilized using Preparative-HPLC to obtain compound D-6 as a white solid (40 mg, 45.6%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.13 (bs, 1H), 7.05 (bs, 1H), 6.72 (s, 2H), 4.68 - 4.65 (m, 1H), 4.25 - 4.03 (m, 4H), 3.97 - 3.91 (m, 1H), 3.82 - 3.70 (m, 6H), 3.65 - 3.55 (m, 11H), 3.52 - 3.47 (m, 1H), 3.39 - 3.36 (m, 1H), 3.31 - 3.24 (m, 2H), 3.12 - 3.07 (m, 4H), 2.97 (s, 1H), 2.91 (s, 3H), 2.64 - 2.56 (m, 2H), 2.55 - 2.50 (m, 2H), 1.82 - 1.75 (m, 3H), 1.66 - 1.54 (m, 6H), 1.46 - 1.42 (m, 9H), 1.37 - 1.35 (m, 2H); EI-MS m/z: 858 [M+H] ⁺ .

Step 4: Preparation of compound D-7

Compound D-6 (40 mg, 0.047 mmol) was dissolved in MC (1 mL) under a nitrogen atmosphere at 0°C. N-hydroxysuccinimide (5.9 mg, 0.051 mmol) and EDC (9.8 mg, 0.051 mmol) were sequentially added, and the mixture was stirred at 0°C for 3 h. After slowly warming to room temperature, the mixture was stirred for 2 h. After completion of the reaction, the mixture was diluted with ACN (2 mL) and distilled water (4 mL), and then separated, purified, and lyophilized using Preparative-HPLC to obtain compound D-7 as a white solid (19.4 mg, 43.6%). EI-MS m/z: 956 [M+H] ^+.

Step 5: Preparation of compound D-8

Under a nitrogen atmosphere at 0℃, compound B-18 (5 mg, 0.006 mmol) was dissolved in 1,4-dioxane (0.5 mL), DMF (0.5 mL), and THF (0.5 mL). Compound D-7 (6.9 mg, 0.007 mmol), DIPEA (1.9 μL, 0.011 mmol), and distilled water (10 μL) were sequentially added, and the mixture was stirred at 0℃ for 30 min, then slowly warmed to room temperature and stirred for 4 h. After completion of the reaction, 2 N hydrochloric acid aqueous solution was slowly added dropwise to adjust the pH of the reaction solution to 4 to 5, and the mixture was diluted with ACN (1 mL) and distilled water (4 mL). The mixture was separated, purified, and lyophilized using Preparative-HPLC to obtain compound D-8 (8.8 mg, 90.7%) as a white solid. EI-MS m/z: 1734 [M+H] ⁺ , 867 1/2 [M+H] ⁺ .

Step 6: Fabrication of the LT398

Compound D-8 (8.8 mg, 0.005 mmol) was dissolved in MC (0.5 mL) and ACN (0.5 mL) at 0°C under a nitrogen atmosphere, and trifluoroacetic acid (0.2 mL) was slowly added. The mixture was stirred at 0°C for 9 h. After completion of the reaction, the mixture was diluted with ACN (1 mL) and distilled water (6 mL), and the residue was separated, purified, and lyophilized using Preparative-HPLC to obtain LT398 as a white solid (2.8 mg, 33.8%). EI-MS m/z: 1634 [M+H] ⁺ , 817 1/2 [M+H] ⁺ .

Example 1. Selection of anti-CLND18.2 antibodies with improved antigen affinity

In order to produce an antibody with improved antigen binding affinity compared to monoclonal 0058-002 (Korean Patent No. 10-2022-0182621), a fully human antibody sequence library with secured sequence diversity was produced centered on the Fv (Fragment variable) sequence of the antibody, and designed to be displayed on phage in the form of scFv (TRO-HUMAN-ANTIBODY-phage display-AFFIMATU Library, TRO-AM Library) based on the reference literature (Smith GP. Science, 228(4705):1315-7 (1985)). CLDN18.2 antigen protein was used to adsorb CLDN18.2-VLPs (#CSB-MP005498HU(A5), CusaBio) onto an immunotube (#NUNC-444202, ThermoFisher), and then treated with the TRO-AM Library phage and reacted at room temperature for 2 hours.

After washing with 1X PBST and 1X PBS (#LB001-02, Welgene), and sequentially treated with 100 mM TAE (#SIAL- 90335, Sigma-Aldrich) and Tris-HCL (pH 7.5) solution, polyphages that specifically bind to the antigen were eluted (panning). Additional panning was performed by increasing the number of washes with 1X PBST and 1X PBS, and then two or more positive monophage clones that ultimately bind to CLDN18.2 were selected using the ELISA method. The selected clones were converted to human IgG1-kappa form, produced, purified, and used in the experiment. At this time, the LALA (L234A, L235A mutant) sequence was introduced into CH2 of the Fc of IgG1 based on the EU numbering, and the K149C sequence was introduced into the CL (Constant light chain) of the kappa light chain based on the Kabat numbering.

The two monoclonal antibodies finally selected (AffiMatu-19, AffiMatu-45) were LS (Light chain Suffling-AffiMatu) clones containing the VH of the 0058-002 clone and the altered VL, and the amino acid sequences of each CDR (Table 1), and the amino acid sequences (Table 2) and base sequences (Table 3) of the heavy and light chains are shown below. In addition, the amino acid sequences of the two antibodies (Zolbetuximab analog, 0058-002) used as anti-CLDN18.2 antibodies are shown in Table 4. The amino acid sequences of the human IgG1 variant (LALA) or the kappa variant (K149C) applied with Thiomab to each antibody are shown in Table 5, and the amino acid sequences and nucleic acid sequences of the heavy and light chains of each antibody are as shown in Tables 6 and 7, respectively.

CLONE NAME HEAVY CHAIN LIGHT CHAIN H-CDR1 H-CDR2 H-CDR3 L-CDR1 L-CDR2 L-CDR3 0058-002 GFTFSSYA
(Sequence number 1) ISGSGGST
(Sequence number 2) ARGLGYYYYGMDV
(Sequence number 3) QTVSSW
(Sequence number 4) AAS
(Sequence number 5) QQYHSFPPT
(Sequence number 31) AffiMatu-19 GFTFSSYA (SEQ ID NO: 1) ISGSGGST
(Sequence number 2) ARGLGYYYYGMDV
(Sequence number 3) QTVSSW
(Sequence number 4) AAS
(Sequence number 5) QQYHSLPPT
(Sequence number 6) AffiMatu-45 GFTFSSYA (SEQ ID NO: 1) ISGSGGST
(Sequence number 2) ARGLGYYYYGMDV
(Sequence number 3) QGISSY
(Sequence number 7) AAS
(Sequence number 5) QQYLSLPVT
(Sequence number 8)

CLONE NAME VARIABLE HEAVY CHAIN VARIABLE LIGHT CHAIN AffiMatu-19 QMQLVESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGLGYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 9) DLVMTQSPSSLSASVGDRVTITCRAS QTVSSW LAWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTEFTLTISSLQPEDFGTYYC QQYHSLPPT FGGGTKVEIK (SEQ ID NO: 10) AffiMatu-45 QMQLVESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGLGYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 9) DLVMTQSPSTLSASVGDKVTITCRAS QGISSY LAWYQQKPGKGPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYC QQYLSLPVT FGGGTKVDIK (SEQ ID NO: 11) UNDERLINE: CDR

CLONE NAME DNA (N-TERMINAL TO C-TERMINAL) AffiMatu-19 VH CAGATGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGC AGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGGTTAGGTTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 34) VL GACCTGGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATAACTTGTCGGGCGAGTCAGACTGTCAGCAGCTGGTTAGCCTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTCTGC AAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGACTTTGGAACTTATTACTGCCAACAGTATCATAGTCTTCCTCCAACATTCGGCGGAGGGACCAAGGTGGAGATCAAG (SEQ ID NO: 20) AffiMatu-45 VH CAGATGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGC AGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGGTTAGGTTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 34) VL GACCTGGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAAAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGGCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGC AAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCTGAAGATATTGCAACATATTACTGTCAACAATATCTTAGTCTCCCGGTAACTTTCGGCGGAGGGACCAAGGTAGATATCAAA (SEQ ID NO: 28)

CLONE NAME VARIABLE HEAVY CHAIN VARIABLE LIGHT CHAIN Zolbetuximab analogs QVQLQQPGAELVRPGASVKLSCKAS GYTFTSYW INWVKQRPGQGLEWIGN IYPSDSYT NYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYC TRSWRGNSFDY WGQGTTLTVSS (SEQ ID NO: 22) DIVMTQSPSSLTVTAGEKVTMSCKSS QSLLNSGNQKNY LTWYQQKPGQPPKLLIY WAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPFT FGSGTKLEIK (SEQ ID NO: 12) 0058-002 QMQLVESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGLGYYYYGMDV WGQGTTVTVSS (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRAS QTVSSW LAWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTEFTLTISSLQPEDFGTYYC QQYHSFPPT FGGGTKVGIK (SEQ ID NO: 13) UNDERLINE: CDR

AMINO ACID (N-TERMINAL TO C-TERMINAL) IgG1-WT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 32) IgG1-LALA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) Kappa LC-WT RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33) Kappa LC-K149C RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW C VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 15) UNDERLINE: A CHANGED AMINO ACID

CLONE NAME AMINO ACID (N-TERMINAL TO C-TERMINAL) Zolbetuximab analogs HEAVY CHAIN QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) LIGHT CHAIN DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEI KRSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17) 0058-002 HEAVY CHAIN QMQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLGYYYYGMDVWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) LIGHT CHAIN DIQMTQSPSSLSASVGDRVTITCRASQTVSSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFGTYYCQQYHSFPPTFGGGTKVGIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) AffiMatu-19 HEAVY CHAIN QMQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLGYYYYGMDVWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) LIGHT CHAIN DLVMTQSPSSLSASVGDRVTITCRASQTVSSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFGTYYCQQYHSLPPTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) AffiMatu-45 HEAVY CHAIN QMQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLGYYYYGMDVWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) LIGHT CHAIN DLVMTQSPSTLSASVGDKVTITCRASQGISSYLAWYQQKPGKGPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYLSLPVTFGGGTKVDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23)

The human Ig sequence similarity of the four anti-CLDN18.2 antibodies was analyzed through the IGBLAST site (https:/www.ncbi.nlm.nih.gov/igblast/) and is shown in Table 8.

CLONE CODE VH (%) VL (%) Germline VH gene Germline VL gene Zolbetuximab analogs 69 71 IGHV1-46*01 IGKV4-1*01 0058-02 99 95 IGHV3-23*04 IGKV1D-16*01 AffiMatu-19 99 93 IGHV3-23*04 IGKV1D-16*01 AffiMatu-45 99 94 IGHV3-23*04 IGKV1-9*03

Example 2. Production, purification, and analysis of anti-CLDN18.2 antibodies

Example 2.1. Production of anti-CLDN18.2 antibodies using a transient expression system.

Each polynucleotide (SEQ ID NO: 26 to SEQ ID NO: 30) encoding the anti-CLDN18.2 antibody was produced by requesting gene synthesis from Bioneer or performing PCR (gene amplification) using oligomers, and then treated with restriction enzymes (NEB) to be cloned into the expression vectors PC3.1-HC heavy chain vector (TriOar) and PC3.1-LC light chain vector (TriOar), respectively.

The expression vectors for the heavy and light chains of each antibody were simultaneously introduced into Expi293F ^TM cell line (#A14527, Thermo) using Thermo's ExpiFectamine ^TM 293 Transfection Kit (#A14524, Thermo) according to the manufacturer's instructions. The cells were cultured in Expi293 ^TM Expression Medium (#A1435101, Thermo) for 5 days under shaking (120 rpm, 8% CO ₂ , 37°C; CO ₂ shaking incubator, N-biotek), and then centrifuged (5,000 rpm, 4°C, 30 min) to obtain the culture supernatant, which was then filtered through a 0.22 um filter.

Example 2.2. Purification of anti-CLDN18.2 antibody using a Protein A column

The culture supernatant obtained by the method of Example 2.2 was purified using a Protein A affinity column (HiTrap ^TM MabSelect SuRe, Cytiva) in an AKTA Pure 25 (Cytiva) or AKTA Avant 150 (Cytiva) instrument, and then the buffer was exchanged through a desalting column (PD-10 Desalting Column, Cytiva). The concentration of the final purified product was quantified by measuring the absorbance at 280 nm using a microquantitative analyzer (Optizen NanoQ, KLAB). The final purified product was subjected to SDS-PAGE under reducing conditions (4-20% Mini-PROTEAN® TGX ^TM Precast Protein Gels, Bio-Rad).

As a result, as shown in Fig. 1, the heavy and light chains of the anti-CLDN18.2 antibody were confirmed at each expected position, and as shown in Table 10, the two selected anti-CLDN18.2 antibodies had excellent productivity in the transient expression system.

Example 2.3. Characterization of anti-CLDN18.2 antibodies using SEC.

A size exclusion chromatography (SEC; TSK gel SuperSW mAbm, TOSOH) column was mounted on an ACQUITY H-Class PLUS Bio System (#176015105, Waters), and each sample (purified product) was diluted in phosphate buffer solution (pH 7.4) and loaded onto the column. The purity was confirmed by analysis at a flow rate of 0.5 mL/min at room temperature for 40 minutes (Fig. 2a, Fig. 2b, and Table 10).

As a result, as shown in Figures 2a and 2b, it was confirmed that the anti-CLDN18.2 antibody was purified to a high purity of 97% to 98% through only the first purification.

Table 9 shows the protein codes of anti-CLDN18.2 antibodies and isotype controls.

PROTEIN CODE CLONE CODE NOTE ZOLBE Zolbetuximab analogs Reference anti-CLDN18.2 antibody TAB01 0058-02 maternal anti-CLDN18.2 antibody TAB07 AffiMatu-19 Selected anti-CLDN18.2 antibodies TAB10 AffiMatu-45 Selected anti-CLDN18.2 antibodies ISO94 Not associated human IgG1-kappa isotype control hIg Not associated(#31154, ThermoFidhser) human IgG isotype control

Example 2.4. Hydrophilicity analysis of anti-CLDN18.2 antibodies using HIC-UPLC

A Butyl-NPR column (#14947, TSK) was mounted on an ACQUITY H-Class PLUS Bio System (#176015105, Waters), and each sample (ZOLBE, TAB01, TAB07, TAB10; 1 mg/mL, 10 uL) was loaded for measurement (HIC; Hydrophobic interaction chromatography-UPLC). 10% Acetonitrile buffer was mixed with each sample from 0% to 100% at a flow rate of 1 mL/min, and the gradient was analyzed at 30°C for 30 min. The results were analyzed using the Waters Empower 3.6.1 version (Waters) program (Fig. 3a, Fig. 3b, and Table 10).

As a result, as shown in Figures 3a and 3b, all four types of antibodies exhibited similar column migration speeds. The column peak of the ZOLBE antibody was clumped together and the detection rate was low. This result is because the ZOLBE antibody has the characteristic of aggregating, and the reason why the ZOLBE antibody was detected relatively quickly with a retention time of 9.5 minutes is thought to be because the column migration was delayed due to the antibody aggregation characteristic.

Example 2.5. Thermostability Analysis of Anti-CLDN18.2 Antibodies Using DSF

Thermal stability of anti-CLDN18.2 antibody was analyzed using differential scanning fluorimetry (DSF).

Specifically, the antibody (preferably 5 uM, total 45 uL) and fluorescent dye (Sypro orange; #S6651, Thermo) (200X, total 5 uL) diluted in DPBS solution were mixed and dispensed into a q-PCR tube (#TLS0801, Biorad). The mixture was reacted in a real-time thermal cycler (CFX Duet Real-Time PCR Detection System, Biorad) by increasing the temperature by 0.5 °C every 30 seconds in the range of 25 °C to 99 °C, and the reaction was completed after 10 minutes of standing reaction at 25 °C. The intensity of fluorescence in the sample solution during the reaction was measured in real time and analyzed using the GraphPad Prism 9 program (Fig. 4 and Table 10).

As a result, as shown in Fig. 4 and Table 10, the melting temperatures of the TAB01 antibody and the two anti-CLDN18.2 antibodies with improved affinity were similar at 67°C to 68°C, while the ZOLBE antibody was relatively low.

Table 10 below shows the molecular characteristics of anti-CLDN18.2 antibodies.

PROTEIN CODE MOLECULAR WEIGHT PI PRODUCTIVITY [mg/L] PURITY
[%] Retention Time [min] THERMAL STABILITY
[Melt temp, ℃] ZOLBE 143846 8.52 608 98 *9.5 66 TAB01 146514 8.32 472 97 12.4 68 TAB07 143892 8.43 647 98 12.4 68 TAB10 143618 8.43 624 98 13.2 68

Example 3. Binding affinity of anti-CLDN18.2 antibodies

Example 3.1. Confirmation of antigen-specific binding affinity of anti-CLDN18.2 antibodies using flow cytometry.

The specific binding affinity of anti-CLDN18.2 antibody to the antigen protein was confirmed through flow cytometry (FC).

Specifically, each cell expressing or not expressing the CLDN18.2 antigen on the cell surface was washed twice (centrifuged (Avanti J-15R, Beckman); 4℃, 1,000 rpm, 3 min), the supernatant was removed, and the cell was suspended in a washing solution (2% FBS/DPBS). The cells suspended in the washing solution at 1 × 10 ⁶ cells/mL were dispensed into 96-deep well plates (#90063, Bioneer) at 0.1 mL each. The antibody was prepared at a concentration twice that of the concentration to be treated in the washing solution (e.g., 15 ug/mL, 1/3-fold, serial dilution), and 0.1 mL each was applied to the dispensed cells. After incubation for 1 hour at 4℃, the cells were washed twice and the washing solution was removed. Secondary antibody (#FI-3000, Vectorlab) was diluted 1:100 in washing solution, 0.1 mL was applied to each cell, and the reaction was performed in the dark at 4°C for 30 minutes.

After the reaction was completed, the cells were washed twice, the washing solution was removed, and the cells were suspended in 0.3 mL of DPBS (#SH30028.02, Hyclone). The cells were maintained at 4℃ in a light-shielded state and analyzed on a flow cytometer (#CytoFlexS, Beckman). The histograms of antibody protein concentrations were obtained using the Kaluza (FC analysis software) program and analyzed using the Graphpad prism 9 program. The cell lines used for flow cytometry are shown in Table 11, and their culture conditions are described in Table 12.

Cell line Cell type Cell source SNU-601 Human gastric carcinoma cell, CLDN18.2-positive #00601, KCLB SNU-620 Human gastric adenocarcinoma cell, CLDN18.2-positive #00620, KCLB PATU8988s Human pancreatic adenocarcinoma cell, CLDN18.2-positive #ACC204, DSMZ KATO III Human gastric carcinoma cell, CLDN18.2-dim #30103, KCLB BxPC3 Human pancreatic adenocarcinoma cell, CLDN18.2-negative, CLDN18.1-postivie Y-Biologics NCI-N87 Human stomach carcinoma, CLDN18.2-negative #60113, KCLB CLDN18.2/HEK293E Human kidney cell, CLDN18.2-overexpressed Y-Biologics HEK293E Human kidney cell, CLDN18.2-negative Y-Biologics CHO-K1 Hamster cell, CLDN18.2-negative

Cell line Growth media SNU-601 RPMI-1640 (#SH30027.01, Hyclone), 25mM HEPES (#15630080, Gibco), 25mM Sodium Bicarbonate (#25080094, Gibco), 10% FBS (#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) SNU-620 RPMI-1640 (#SH30027.01, Hyclone), 25mM HEPES (#15630080, Gibco), 25mM Sodium Bicarbonate (#25080094, Gibco), 10% FBS (#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) PATU8988s DMEM with High Glucose(#SH30243.01, Hyclone), 5% FBS(#SH30919.03, Hyclone), 5% Horse Serum(#16050122, Gibco), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) KATO III RPMI-1640 (#SH30027.01, Hyclone), 25mM HEPES (#15630080, Gibco), 25mM Sodium Bicarbonate (#25080094, Gibco), 10% FBS (#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) BxPC3 RPMI-1640(#SH30027.01, Hyclone), 10% FBS(#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) NCI-N87 RPMI-1640 (#A10491-01, Gibco), 10% FBS(#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) CLDN18.2/HEK293E DMEM with High Glucose(#SH30243.01, Hyclone) , 10% FBS(#SH30919.03, Hyclone) , 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone), 400 ug/ml G418(#10131-027, Gibco) HEK293E DMEM with High Glucose(#SH30243.01, Hyclone) , 10% FBS(#SH30919.03, Hyclone) , 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone) CHO-K1 RPMI-1640 (#SH30027.01, Hyclone), 25mM HEPES (#15630080, Gibco), 25mM Sodium Bicarbonate (#25080094, Gibco), 10% FBS (#SH30919.03, Hyclone), 1% Antibiotic Antimycotic Solution(#SV30079.01, Hyclone)

As a result, as shown in FIGS. 5a to 5e, no binding of anti-CLDN18.2 antibodies was observed in HEK293E cells that do not express CLDN18.2 antigen protein on the cell surface (FIG. 5a), whereas binding to the antigen was observed in CLDN18.2/HEK293E cells in which CLDN18.2 was overexpressed in a concentration-dependent manner according to the treated antibody (FIG. 5b). In addition, antibody binding was observed in a concentration-dependent manner in human gastric cancer cell lines (SNU-601 cells) and human pancreatic cancer cell lines (PATU8988s cells) in which CLDN18.2 is endogenously expressed. In particular, the TAB01 antibody and the two antibodies, which are specific examples of the present invention, had superior antigen binding affinity compared to the ZOLBE antibody (FIGS. 5c and 5d).

In the above results, it was confirmed that the TAB01 antibody and the two selected antibodies (TAB07 antibody, TAB10 antibody) specifically bind only to cells expressing the CLDN18.2 antigen. At this time, in cells in which the CLDN18.2 antigen protein was overexpressed, the binding affinity was similar to that of the ZOLBE antibody. In addition, in cells in which CLDN18.2 was expressed at an intermediate or low level, the binding affinity of the ZOLBE antibody was weak, whereas the TAB01 antibody, TAB07 antibody, and TAB10 antibody showed excellent binding affinity, and in particular, the binding affinity of the TAB07 antibody and TAB10 antibody was the highest.

Example 3.2. Confirmation of selective binding affinity through flow cytometry

The selective binding ability of anti-CLDN18.2 antibodies to BxPC-3 cells, which are CLDN18.2-negative cells or CLDN18.1-positive cells, was confirmed in the same manner as in Example 3.1.

At this time, anti-CLDN18 (# CSB-RA005498A2HU, CusaBio) antibody was used as a positive control. The positive control is an antibody that binds to CLDN18.1.

As a result, as shown in Fig. 5e, antigen-antibody binding was observed in the positive control group in BxPC3 cells, but no antibody binding was observed for the TAB01 antibody, TAB07 antibody, and TAB10 antibody.

Through the above results, it was confirmed that TAB01 antibody, TAB07 antibody, and TAB10 antibody do not bind to CLDN18.1, but selectively bind only to CLDN18.2.

Example 4. Analysis of CLDN18.2 antibody internalization using confocal microscopy.

The surface antigen-dependent internalization ability of anti-CLDN18.2 antibody was confirmed using a confocal microscope.

Specifically, 12 mm coverslips (#0111520, Superior MARIENFELD) coated with Poly-L-lysine (#P4707, Sigma) were placed in 24-well plates (#353047, Falcon), and CLDN18.2/HEK293E cells were seeded with 2 × 10 ⁴ cells/0.5 mL per well, and cultured at 5% CO ₂ and 37°C for 24 h. Afterwards, each well was washed twice with DPBS (#LB001-02, Welgen), and serum-free medium (DMEM/1% Antibiotic/Antimycotic Solution/400 ug/mL G418) was added, and cultured for 16 h. Then, anti-CLDN18.2 antibodies were treated at each concentration in cold DPBS (0.5 mL) and allowed to stand for 1 h at 4°C.

Each well was washed twice with cold DPBS, treated with cell culture medium containing 10 mM NH ₄ Cl (#12125-02-9, Daejeong), and incubated at 5% CO ₂ and 37°C for 24 h. Each well was washed twice with DPBS, treated with 4% paraformaldehyde (#15710, Electron Microscopy Sciences (16% Paraformaldehyde)), and incubated for 10 min at room temperature. Each well was washed twice with DPBS containing 10% FBS, and treated with anti-LAMP1 antibody (#ab24170, Abcam) diluted in reaction solution (0.5% saponin (#A18820.22, Alfa Aesar)/PBS/10% FBS) and incubated for 1 h at room temperature. Afterwards, each well was washed twice with DPBS containing 10% FBS, and Alexa Fluor 488 anti-human IgG (H+L) antibody (#709-545-149, Jackson ImmunoResearch) and Cy™3 AffiniPure anti-rabbit IgG antibody (#111-165-144, Jackson ImmunoResearch) diluted in reaction solution were added and reacted at room temperature for 1 hour. Each well was washed with DPBS containing 10% FBS, mounted with mounting solution containing DAPI (ProLong™ Diamond Antifade Mountant with DAPI, #P36971, Invitrogen), and cellular internalization was observed using a confocal microscope (#LSM900, ZEIZZ) (Fig. 6).

As a result, no cell internalization was observed when IgG was treated (Fig. 6a). In contrast, TAB01 antibodies (Fig. 6b), TAB07 antibodies (Fig. 6c), and TAB10 antibodies (Fig. 6d) were observed on the cell surface at 0 h (green fluorescence) or at the same location as LAMP-1 (red fluorescence), an intracellular lysosomal marker, at 24 h.

The above results confirmed that TAB01 antibody, TAB07 antibody, and TAB10 antibody could be internalized into cells in which CLDN18.2 was expressed on the cell surface.

Example 5. Preparation and Analysis of Anti-CLDN18.2 Antibody-Drug Conjugate

Example 5.1. Conjugation and purification of anti-CLDN18.2 antibody and drug.

Antibody-drug conjugates for each compound were prepared with reference to the methods described in Nature Biotechnology(2008)26:925-932; Bioconjugate Chem.(2013)24:1256-1263; Bioconjugate Chem.(2016)27:1324-1331; Bioconjugate Chem.(2014)25:460-469.

The linker-payload conjugates used were LT409 of Synthesis Example 1, LT398 of Synthesis Example 2, and the conjugates disclosed in International Publication No. WO 2024/005460 A1 (compounds B-35, B-53, B-52, B-17, B-19, and B-50) as shown in Table 14 below.

LT397 and LT399 were manufactured by changing the starting material Mal-PEG2-acid to Mal-PEG5-acid in the manufacture of LT380 and LT384, respectively.

Specifically, antibody-drug conjugates were prepared by reducing antibody molecules by adding a reducing agent and then treating the linker-payload conjugate at a molar concentration of 10 times or more. At this time, the bound antibody-drug conjugate was purified by removing the remaining unbound linker-payload conjugate using a PD-10 (#17-0851-01, Cytiva) column. At this time, the bound antibody-drug conjugate was purified by removing the remaining unbound linker-drug using a PD-10 (#17-0851-01, Cytiva) column, and the final purified yield (conjugation yield) is shown in Table 13 below. In addition, a linker which is an embodiment of the present invention is shown in Table 14.

As a result, as shown in Table 13, the yield of the antibody-drug conjugate including the ZOLBE antibody was very low due to the characteristic of the antibody to aggregate, and the final yield of the antibody-drug conjugate including the TAB01 antibody, TAB07 antibody, or TAB10 antibody was excellent, being around 56% to about 80%.

Example 5.2. Analysis of anti-CLDN18.2 antibody-drug conjugation rate

The drug-antibody ratio (DAR) was measured using an ACQUITY H-Class PLUS Bio System (#176015105, Waters) equipped with a PLRP-S column for biomolecules (#PL1912-3802, Agilent).

Specifically, each sample (1 mg/mL, 10 uL) was directly loaded onto the column (RP-UPLC) or loaded after treating with DTT at a concentration of 5 mM to 50 mM for 30 minutes and then measured (rRP-UPLC). Acetonitrile was mixed with each sample at a concentration of 30% to 50% at a flow rate of 1 mL/min, and analyzed by increasing and decreasing the temperature at 80°C for 20 minutes. The results were analyzed using the Waters Empower 3.6.1 version (Waters) program.

The antibody-drug conjugation rate can be confirmed through the chromatograms in Table 13 above and FIGS. 7A to 7K. Even when various linkers and various payloads were prepared using various conjugation methods (position-specific or position-nonspecific) for TAB01 to TAB07 and TAB10 antibodies with improved affinity, it was confirmed that the desired payload conjugation rate was consistently conjugated at about 80% or more.

Example 5.3. Size Variant Analysis of Anti-CLDN18.2 Antibody-Drug Conjugate

Analysis of size variants of antibody-drug conjugates was performed in the same manner as in Example 2.3.

As shown in Table 13 above and Figures 8a to 8g, the finally obtained antibody-drug conjugate exhibited a high purity of 90% or more.

Example 5.4. Confirmation of the hydrophilicity of the anti-CLDN18.2 antibody-drug conjugate.

The hydrophilicity of an anti-CLDN18.2 antibody-drug conjugate (TAB07.409.1), which is a specific example of the present invention, and TAB07.121.1 (GGFG-DXD), which has an anti-CLDN18.2 antibody and a clinically proven Enhertu linker, was compared. The hydrophilicity was determined in the same manner as in Example 2.4.

As a result, as shown in Fig. 9, it was confirmed that TAB07.409.1 exhibited superior hydrophilicity compared to TAB07.121.1.

Example 6. In vitro cytotoxicity of anti-CLDN18.2 antibody-drug conjugate

In vitro cytotoxicity of the anti-CLDN18.2 antibody-drug conjugate was confirmed using the tumor cell lines and cell culture media in Tables 11 and 12 above.

Specifically, each tumor cell line was seeded into a 96-well plate (#83.3924, Sarstedt) at 1 × 10 ³ to 3 × 10 ³ cells per well and cultured for 24 hours. Thereafter, each well was treated with an antibody-drug conjugate, which is an embodiment of the present invention, and cultured for 96 to 144 hours. After completion of the culture, each well was treated with 20 uL of MTS solution (#G3581, Promega) and reacted in a CO ₂ incubator for 4 hours. The absorbance of the reaction solution was measured at a wavelength of 490 nm using a spectrometer (#GM3000, Promega).

The above results were analyzed using the GraphPad Prism 9 program and are shown in Fig. 9. At this time, Figs. 10a to 10f and Fig. 10h are the results of processing an antibody-drug conjugate in which a linker-payload is position-specifically conjugated to an antibody, and Fig. 10g is the result of processing an antibody-drug conjugate in which an antibody is position-nonspecifically conjugated.

Figures 10a and 10b show the cytotoxicity of an antibody-drug conjugate comprising four MMAE microtubule inhibitors conjugated to an anti-CLDN18.2 antibody (4 days). In cell lines expressing the CLDN18.2 antigen protein on their surface, TAB01.348 exhibited superior cytotoxicity compared to ZOLBE.348. Neither antibody-drug conjugate exhibited cytotoxicity in HEK293E cells, which are CLDN18.2-negative cells.

Figure 10c shows the results of cytotoxicity testing of an antibody-drug conjugate comprising two microtubule inhibitors, MMAE or MMAF, and a topoisomerase inhibitor, DXD or Exatecan, conjugated to the TAB01 antibody using a clinically proven linker or a linker system, which is an embodiment of the present invention, after treatment of a human gastric cancer cell line (SNU-601 cells). The antibody-drug conjugate produced using the linker system, which is an embodiment of the present invention, exhibited superior cytotoxicity in SNU-601 cells.

Figure 10d shows the results of cytotoxicity testing of antibody-drug conjugates conjugated to TAB01 antibody using a clinically proven linker or a linker system, which is an embodiment of the present invention, on a cell line (CLDN18.2/HEK293E cells) engineered to overexpress CLDN18.2 on the cell surface. Compared to the antibody-drug conjugate treatment group with a clinically proven linker, the antibody-drug conjugate to which the linker system, which is an embodiment of the present invention, was applied exhibited higher cytotoxicity. In particular, the cytotoxicity efficacy of TAB01.380 (LT380-Exatecan) conjugated with a linker, which is an embodiment of the present invention, was significantly superior compared to TAB01.121 (GGFG-DXD), and the efficacy of TAB01.347 (LT347-MMAF) conjugated with a truncated linker, which is an embodiment of the present invention, was superior compared to the non-cleavable linker (TAB01.122, LT122-MMAF).

Figure 10e shows the results of cytotoxicity testing of TAB01.399 (LT399-Exatecan), which is a specific example of the present invention and is obtained by conjugating Exatecan to TAB01, TAB07, or TAB10 antibodies using a linker system, in a cell line overexpressing CLDN18.2 (CLDN18.2/HEK293E cells) or a gastric cancer cell line with intermediate/low expression (SNU-601 cells). Compared to ZOLBE.399, the cytotoxicity of TAB01.399 was superior in both cell lines.

Figure 10f shows the results of confirming the cytotoxicity of four types of antibody-drug conjugates in human pancreatic cancer cell line (PATU8988s cells).

Figure 10g shows the results of cytotoxicity tests performed on human gastric cancer cell lines (SNU-601 cells) after two antibody-drug conjugates, each of which is conjugated to six Exatecans via a linker system, which is an embodiment of the present invention, were treated with the conjugates without specificity for the location of the antibodies. Both antibody-drug conjugates exhibited excellent cytotoxicity on SNU-601 cells, which expressed the CLDN18.2 antigen at intermediate/low levels on the cell surface.

Figure 10h shows the results of cytotoxicity testing of TAB07.409.1 (LT409-Exatecan), which is an antibody conjugated to Exatecan using a linker system, which is an embodiment of the present invention, on a cell line (-CLDN18.2, CHO-K1 cells) in which CLDN18.2 is not overexpressed on the cell surface or on a cell line (CLDN18.2/HEK293E cells) in which CLDN18.2 is overexpressed. TAB07.409.1 did not exhibit cytotoxicity on a cell line (CHO-K1 cells) in which CLDN18.2 was not overexpressed on the cell surface. On the other hand, it exhibited excellent cytotoxicity on a cell line (CLDN18.2/HEK293E cells) in which CLDN18.2 was overexpressed on the cell surface. Additionally, TAB07.409.1 showed significantly superior cytotoxicity compared to TAB07.121.1 (GGFG-DXD), which was introduced with the clinically proven Enhertu linker.

The above results demonstrate that when a microtubule inhibitor or topoisomerase inhibitor drug was conjugated to a specific example of the present invention via a linker system, TAB01 antibody, TAB07 antibody, or TAB10 antibody, cytotoxicity was observed in cells expressing CLDN18.2 on the cell surface (at low/medium/high levels). The cytotoxicity was confirmed to be superior to that of a drug conjugate of a clinically approved Zolbetuximab analog or an anti-CLDN18.2 antibody (TAB07)-drug conjugate (TAB07.121.1) introduced with a linker of Enhertu.

Example 7. In vivo confirmation of anticancer activity of anti-CLDN18.2 antibody-drug conjugate

The anticancer activity of antibody-drug conjugates in which each payload (MMAE, Belotecan, Exatecan) is conjugated to a specific example of the present invention, a linker system, to TAB01 antibody or TAB07 antibody in vivo was confirmed using a xenograft tumor mouse model by requesting a research service organization (DTNCRO, Kipron Bio).

Specifically, human-derived tumor cell lines were mixed with Matrigel in equal amounts and implanted subcutaneously into the flanks of athymic nude or BALB/c nude mice. The tumor volume was calculated as in <Mathematical Formula I>, and on the day of administration, mice with tumor volumes that met the condition were selected and divided into groups using the paired-matching method. Test substances (TAB01.348, TAB01.383, TAB01.384, TAB07.409.1) were administered as a single intravenous (I.V.) injection. At this time, an antibody-drug conjugate (ZOLBE.348) in which MMAE was conjugated to a Zolbetuximab analog or an antibody-drug conjugate (TAB07.121.1, GGFG-DXD) in which a linker of Enhertu was introduced into the TAB07 antibody were used as positive controls. After administration of the test substance, tumor volume and clinical symptoms were measured twice a week. Tumor growth inhibition (TGI) was calculated using <Mathematical Formula II>.

<Mathematical Formula I>

Tumor volume (㎣) = (a × 2b) / 2

Here, a is the short axis and b is the long axis.

<Mathematical Formula II>

Tumor growth inhibition rate (TGI: IR(%)) = (1-T/C) × 100

At this time, T is the average value of the tumor weight of the test substance administration group and the positive control group, and C is the tumor weight of the negative control group.

At the end of the observation period, each subject was euthanized, and tumor tissue was removed, weighed, and imaged. All data obtained during the test period were recorded in an Excel spreadsheet, and the results were analyzed and presented in Figure 11.

Figure 11a shows the results of confirming the anticancer effects of TAB01.348, TAB01.383, and TAB01.384 in a tumor mouse model transplanted with a human gastric cancer cell line (SNU-601 cells) expressing intermediate/low levels of CLDN18.2. All three antibody-drug conjugates exhibited excellent anticancer efficacy. TAB01.348 exhibited superior anticancer efficacy compared to ZOLBE.348, which was conjugated with the same anticancer drug. In particular, in the TAB01.383 and TAB01.384 administration groups, tumors disappeared in four out of five experimental animals, confirming the superior anticancer efficacy of TAB01.383 and TAB01.384. At this time, no pathological abnormalities, including body weight, were observed during the test period.

Figure 11b shows the results of confirming the anticancer efficacy of TAB01.348, TAB01.383, and TAB01.384 in a tumor mouse model transplanted with a human pancreatic cancer cell line (PATU8988s cells) expressing intermediate/low levels of CLDN18.2. ZOLBE.348 and the three antibody-drug conjugates above all exhibited excellent anticancer efficacy.

Figure 11c shows the results of confirming the anticancer effects of TAB07.409.1 or TAB07.121.1 in a tumor mouse model transplanted with a human gastric cancer cell line (SNU-601 cells) expressing intermediate/low levels of CLDN18.2. TAB07.409.1 exhibited superior anticancer effects compared to TAB07.121.1 (GGFG-DXD). No pathological abnormalities, including body weight, were observed during the test period.

Through the above results, it was confirmed that when a microtubule inhibitor or a topoisomerase inhibitor drug was conjugated to a specific example of the present invention, the TAB01 antibody, TAB07 antibody, or TAB10 antibody, through a linker system of the present invention, the antibody-drug conjugate can exhibit anticancer efficacy against tumor cells expressing CLDN18.2 at low/intermediate levels. In particular, it was confirmed that it exhibited anticancer efficacy similar to or superior to that of a drug conjugate of a clinically approved Zolbetuximab analog in not only gastric cancer but also pancreatic cancer models. In addition, in the case of the TAB07 antibody, it was confirmed that the antibody-drug conjugate (TAB07.409.1) in which an anticancer drug was linked through a linker of the present invention exhibited superior anticancer activity compared to the antibody-drug conjugate (TAB07.121.1, GGFG-DXD) in which an Enhertu linker was introduced.

The above results demonstrate that TAB01 antibody, TAB07 antibody, and TAB10 antibody can be used as cancer therapeutics by forming antibody-drug conjugates with various payloads through a linker system, which is an example of a truncated form of the present invention, and confirm the applicability as an antibody-drug therapeutic for pancreatic cancer, including gastric cancer.

Example 8. Stability of anti-CLDN18.2 antibody-drug conjugate

Example 8.1. Confirmation of the stability of anti-CLDN18.2 antibody-drug conjugate in plasma.

Stability of anti-CLDN18.2 antibody-drug conjugate in rat plasma or human plasma Confirmed.

Specifically, TAB07.409.1 (1 mg/mL), which is an example of the anti-CLDN18.2 antibody-drug conjugate of the present invention, was treated with rat plasma (Biochemed, 031-APSC-PMG) or human plasma (BBI solutions, SF505-7), mixed, and reacted in a 37°C incubator. At this time, a certain amount was collected before the reaction, at the start of the reaction (0), and on days 1, 2, 4, and 7 from the start of the reaction, and the residual amounts of tAb (total antibody) and acDrug (Active analyte of ADC) were quantitatively analyzed using a TripleTOF 5600 mass spectrometer.

As a result, as shown in Fig. 12, it was confirmed that TAB07.409.1 was stable for a long time in rat plasma and human plasma.

Example 8.2. Analysis of drug-antibody ratio of anti-CLDN18.2 antibody-drug conjugate

For the plasma samples obtained by the method of Example 8.1, the drug antibody ratio (DAR) of TAB07.409.1 was confirmed using rat plasma (Biochemed, 031-APSC-PMG) and human plasma (BBI solutions, SF505-7). At this time, the change in DAR of TAB07.409.1 was analyzed based on the concentration on day 0.

As a result, as shown in Fig. 13, it was confirmed that TAB07.409.1 was stable and maintained DAR in plasma for a long time.

Example 8.3. Pharmacodynamics of anti-CLDN18.2 antibody-drug conjugates

The pharmacokinetics of the anti-CLDN18.2 antibody-drug conjugate were confirmed by the following method.

Specifically, TAB07.409.1 was administered intravenously as a single dose of 2.5 mg/kg to female rats (week-old). Then, 0.4 mL of blood was collected from the jugular vein using a 1 mL (25 Gauge) syringe treated with heparin (85 IU/mL, 35 uL) on days 0.02, 0.17, 0.33, 1, 2, 4, 7, 11, 21, and 28. The blood collected as described above was injected into a microtube, rolled in a mixer for several minutes, and centrifuged at 14,000 rpm for 5 minutes to separate the plasma. The separated plasma was placed in a microtube and stored in an ultra-low temperature freezer until analysis, and the test substance in the plasma was analyzed using a TripleTOF 5600 mass spectrometer.

As a result, as shown in Fig. 14, it was confirmed that TAB07.409.1 exists very stably in the blood of a living organism for a long time.

Claims (25)

A heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or An anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In the first paragraph, The antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; or An anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. A polynucleotide encoding the antibody of claim 1 or an antigen-binding fragment thereof. A vector comprising the polynucleotide of claim 3. Cells transformed with the vector of Article 4. i) a step of culturing the transformed cell of clause 5; and ii) A method for producing an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, comprising the step of obtaining an antibody or an antigen-binding fragment thereof from a culture medium of the above cells. A pharmaceutical composition for preventing or treating cancer, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. In paragraph 7, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. An anti-CLDN18.2 antibody or an antigen-binding fragment thereof comprising the following heavy and light chain variable regions; and an antibody-drug conjugate comprising an anticancer agent: A heavy chain variable region comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising an amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 31; A heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or An anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In paragraph 9, The antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 13; A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; or An antibody-drug conjugate comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. In paragraph 9, The above anticancer drugs include Methotrexate, Taxol, L-asparaginase, Mercaptopurine, Thioguanine, Hydroxyurea, Cytarabine, Cyclophosphamide, Ifosfamide, Nitrosourea, Cisplatin, Carboplatin, Mitomycin, Dacarbazine, Procarbazine, Topotecan, Nitrogen mustard, Cytoxan, Etoposide, 5-fluorouracil, BCNU (Bis-chloroethylnitrosourea), Irinotecan, Camptothecin, Exatecan, Belotecan, Bleomycin, Doxorubicin, Idarubicin, Daunorubicin, Dactinomycin, Plicamycin, Mitoxantrone, Asparaginase, Vinblastine, Vincristine, Vinorelbine, Paclitaxel, Docetaxel, Chlorambucil, Melphalan, Carmustine, Lomustine Busulfan, Treosulfan, Decarbazine, Etoposide, Teniposide, Topotecan, 9-aminocamptothecin, Crisnatol, Mitomycin C, Trimetrexate, Mycophenolic acid, Tiazofurin, Ribavirin, EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), Hydroxyurea, Deferoxamine, Floxuridine, Doxifluridine, Raltitrexed Cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, Any one or more selected from the group consisting of Gemcitabine, Velcade, Revamid, Thalamid, Lovastatin, 1-methyl-4-phenylpyridinium ion, Staurosporine, Actinomycin D, Dactinomycin, Bleomycin A2, Bleomycin B2, Peplomycin, Epirubicin, Prarubicin, Zorubicin, Mitoxantrone, Verapamil, and Thapsigargin. Antibody-drug conjugate. In paragraph 9, An antibody-drug conjugate, wherein the anti-CLDN18.2 antibody or antigen-binding fragment and the anticancer agent are linked via a linker. In Article 12, An antibody-drug conjugate having a structure represented by structural formula I or structural formula II: <Structural formula I> Ab-[LD] _n <Structural formula II> Ab-[L'-D ₂ ] _n At this time, in the structural formulas I and II, Ab is the antibody of the first clause or an antigen-binding fragment thereof, L and L' are each independently a linker or a direct bond, D is an anticancer drug, n is a real number between 1 and 10. In Article 13, An antibody-drug conjugate, wherein the linker is selected from the group consisting of a non-cleavable linker, a cleavable linker, and a combination thereof. In Article 13, An antibody-drug conjugate, wherein the non-cleavable linker is a maleimide linker. In Article 14, An antibody-drug conjugate, wherein the cleavable linker is a chemically cleavable linker or an enzymatically cleavable linker. In Article 16, An antibody-drug conjugate, wherein the enzyme-cleavable linker is a peptide-based linker or a specific enzyme-based linker. In Article 16, An antibody-drug conjugate, wherein the cleavable linker is at least one selected from the group consisting of a hydrazone linker, an ester linker, a disulfide linker, a valine-citruline linker, a valine-alanine linker, a phenylalanine-glycine linker, a β-galactoside linker, a β-glucuronide linker, and a phosphodiester linker. In Article 14, The antibody-drug conjugate is any one selected from the following <Chemical Formula VI>: <Chemical Formula VI> In the above chemical formula VI, The above mAb is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, The above m5, m6, m9, m10, m11, m12, n9, n10, n13, n14 and n15 are each independently integers from 1 to 10, wherein R ^d3 , R ^d5 and R ^d6 are each independently H or C _1-8 alkyl, wherein Z ¹ is a heteroatom selected from NR ³ , O, S and Se, R ³ is H or C _1-8 hydrocarbyl, The above D is an anticancer agent, The above n is a real number between 1 and 10. In Article 14, The antibody-drug conjugate is any one selected from the following <Chemical Formula VII>: <Chemical Formula VII> In the above chemical formula VII, The above mAb is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, The above D is an anticancer drug, and the anticancer drugs may be the same or different. wherein Z ¹ is a heteroatom selected from NR ³ , O, S and Se, R ³ is H or C _1-8 hydrocarbyl, The above q1 to q3 are each independently an integer from 0 to 10, and the above q4 is an integer from 1 to 10, wherein R ^d1 , R ^d5 and R ^e are each independently H or C _1-8 alkyl, The above n1, n3, n4, n8, n14 and n15, m2, m8 and m10 are each independently integers from 1 to 8, The above n is a real number between 1 and 10. In paragraph 9, An antibody-drug conjugate wherein the anti-CLDN18.2 antibody or an antigen-binding fragment thereof is conjugated to the following linker-payload conjugate: . A pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate of claim 9 as an active ingredient. In paragraph 22, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Use of the antibody or antigen-binding fragment thereof of claim 1, or the antibody-drug conjugate of claim 9, for the prevention or treatment of cancer. A method for preventing or treating cancer, comprising administering to a subject the antibody or antigen-binding fragment thereof of claim 1, or the antibody-drug conjugate of claim 9.