Antibody-drug conjugate comprising CT83 antigen-specific antibody or antigen-binding fragment thereof

Abstract

The present invention relates to an antibody-drug conjugate (ADC) targeting a CT83 (KK-LC-1) antigen. The ADC can induce concentration-dependent apoptosis in CT83-expressing cancer cells and exhibits nanomolar (nM)-level binding affinity, and thus can exhibit excellent target selectivity and anti-tumor efficacy. Therefore, the ADC according to the present invention may be applied as an effective target-based anticancer therapeutic platform that can be utilized in the treatment of various CT83-expressing cancer cells.

Description

Antibody-drug conjugate comprising a CT83 antigen-specific antibody or an antigen-binding fragment thereof

[0001] The present invention relates to an antibody-drug conjugate (ADC) in which a drug is conjugated to an antibody targeting the CT83 (Cancer / Testis Antigen 83) antigen or an antigen-binding fragment thereof.

[0002]

[0003] Antibody-drug conjugates (ADCs) are a next-generation precision medicine technology that combines the target specificity of antibodies with the potent anticancer effects of cytotoxic drugs. They are attracting attention as a therapeutic strategy capable of selectively killing cancer cells while minimizing damage to normal tissues. Conventional cytotoxic anticancer drugs have problems such as causing severe side effects due to non-specific action and limited therapeutic efficiency due to low tumor selectivity. Accordingly, ADC technology has been developed to selectively deliver drugs into cells by conjugating them to antibodies that accurately target antigens overexpressed in cancer cells, and this is recently expanding as a treatment option for various solid tumors and hematological cancers.

[0004] Meanwhile, CT83 (Cancer / Testis Antigen 83, KK-LC-1) is known as a cancer / testis antigen that exhibits high expression in various solid tumors and is reported to be involved in tumor proliferation and metastasis. In particular, CT83 expression is confirmed in various cancer types, including breast cancer, lung cancer, gastric cancer, and cervical cancer; since it is expressed only restrictively in normal tissues, it is suitable for use as a cancer cell-specific target.

[0005] The key to the development of ADC therapeutics is securing antibodies with high selectivity and internalization capabilities for target antigens, which enable the effective delivery of drugs into tumor cells. However, despite the known potential of CT83 as a target for solid tumors, antibodies targeting the CT83 antigen are currently very limited, and no CT83-based ADC therapeutic has yet been developed. Furthermore, since the structural characteristics of CT83 and the epitope information that antibodies can recognize have not been sufficiently elucidated, securing the antibodies necessary for ADC development remains a technical challenge.

[0006] Therefore, the development of CT83-targeted antibody-drug conjugates that recognize the CT83 antigen with high specificity and affinity and enable selective drug delivery through drug conjugation represents an important technical necessity in the field of solid tumor therapeutics.

[0007]

[0008] The objective of the present invention is to provide an antibody or an antigen-binding fragment thereof capable of recognizing the CT83 (Cancer / Testis Antigen 83) antigen with high specificity and affinity, and to provide an antibody-drug conjugate (ADC) capable of selectively delivering a drug to CT83-expressing cancer cells based thereon. Specifically, the main objective of the present invention is to overcome the technical limitations in which appropriate target antibodies were not previously secured despite the CT83 antigen being overexpressed in various solid tumors, and to establish a novel antibody-based ADC technology designed to enable drug delivery by inducing efficient internalization into the cell after antibody binding.

[0009] Furthermore, the present invention has an additional objective of providing a precision anticancer treatment technology capable of expanding therapeutic applicability across CT83-expressing cancers, such as breast cancer, lung cancer, gastric cancer, and cervical cancer, by providing an ADC composition capable of applying various cytotoxic drugs, linkers, and conjugation strategies based on an antibody or antigen-binding fragment targeting CT83, thereby presenting new treatment options for cancers that were difficult to treat with existing therapies.

[0010] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0011]

[0012] To solve the above technical problem, the present invention provides a novel antibody or an antigen-binding fragment thereof that specifically binds to the CT83 antigen, and an antibody-drug conjugate (ADC) capable of selectively delivering a drug to CT83-expressing cancer cells by conjugating a cytotoxic drug to the antibody or fragment.

[0013] According to one embodiment of the present invention, the antibody or antigen binding fragment is composed of a heavy chain variable region including heavy chain CDR1 of SEQ ID NO. 7, heavy chain CDR2 of SEQ ID NO. 8, and heavy chain CDR3 of SEQ ID NO. 9, and a light chain variable region including light chain CDR1 of SEQ ID NO. 10, light chain CDR2 of SEQ ID NO. 11, and light chain CDR3 of SEQ ID NO. 12, and can be designed to recognize the CT83 antigen with high specificity and affinity.

[0014] According to another embodiment of the present invention, the antibody or antigen-binding fragment may be optimized to recognize the epitopes of SEQ ID NOs. 17 and 18.

[0015] According to another embodiment of the present invention, the antibody or its antigen-binding fragment may be scFv, and the drug may comprise one or more selected from the group consisting of toxins, mitotic inhibitors, apoptotic inducers, anticancer chemotherapy agents and antitumor agents, and more specifically may comprise one or more selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), meitansinoid (DM1), caliceamicin, duocamycin, and anthracycline.

[0016] According to another embodiment of the present invention, the antibody or its antigen-binding fragment and the drug are conjugated through a linker, and the linker may have the structure of Formula 1.

[0017] <Chemical Formula 1>

[0018]

[0019] According to another embodiment of the present invention, the present invention may provide a composition for the prevention or treatment of cancer comprising the antibody-drug conjugate as an active ingredient, or a method for the prevention or treatment of cancer comprising administering an effective amount of the antibody-drug conjugate to a cancer patient, wherein the cancer may be cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer. It may include one or more selected from the group consisting of cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, multiple myeloid blood cancer, and solid tumors.

[0020]

[0021] According to the present invention, by providing an antibody-drug conjugate in which a drug is conjugated based on an antibody targeting the CT83 antigen or an antigen-binding fragment thereof, the problems of non-specific toxicity and low selectivity of existing anticancer drugs can be effectively overcome. The antibody presented in the present invention includes CDR combinations defined by SEQ ID NOs. 7 to 12, thereby recognizing the CT83 antigen with high affinity and specificity. These binding characteristics can maximize drug delivery efficiency by selectively inducing internalization in cancer cells that highly express CT83. Accordingly, the antibody-drug conjugate of the present invention can exhibit excellent cytotoxic effects on CT83-positive tumor cells while minimizing unintentional drug delivery to normal cells.

[0022] Furthermore, the antibody or antigen-binding fragment of the present invention can also be implemented in the form of scFv, enabling excellent tissue accessibility and internalization capabilities even in environments where penetration is difficult, such as solid tumor tissues. This improves the tissue permeability issues pointed out as limitations in existing antibody-based therapies and significantly enhances efficacy in the treatment of solid tumors.

[0023] Furthermore, the antibody-drug conjugate of the present invention is applicable not only to various solid tumors expressing CT83, such as cervical cancer, breast cancer, lung cancer, colorectal cancer, ovarian cancer, and bladder cancer, but also to some hematological cancers, thereby providing broad clinical applicability capable of treating multiple types of cancer based on a single platform. Due to these technical characteristics, the present invention can be utilized as a highly useful therapeutic technology that expands new areas of anticancer treatment while resolving the target limitation issues of existing antibody-drug conjugates.

[0024]

[0025] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims.

[0026]

[0027] Figure 1 shows the ELISA results of 400 anti-CT83 monoclonal antibody candidates.

[0028] Figure 2 shows the ELISA results of SKAI-48, which was selected as an antibody candidate of the present invention.

[0029] Figure 3 shows the microarray results confirming binding with the antigen for epitope mapping.

[0030] Figure 4 shows the expression levels of KK-LC-1 protein in HeLa cells or HT-29 cells, measured with invitrogen’s CT83 monoclonal antibody (CL4762).

[0031] Figure 5 confirms whether SKAI-48, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.

[0032] Figure 6 shows some information about the epitope of the antibody SKAI-48 of the present invention. The top of Figure 6 shows information on the sequence, length, mass, isoelectric point, charge, hydrophobicity value, and extinction coefficient of the epitope; the middle of Figure 6 shows the structure of the epitope; and the bottom of Figure 6 shows the sequence, sequence number, length, and hydrophobicity index for the amino acids forming the epitope sequence.

[0033] Figure 7 illustrates the sequence of an epitope of the antibody SKAI-48 of the present invention. Figure 7a shows a partial sequence position of the epitope of the antibody SKAI-48 of the present invention, Figure 7b shows another partial sequence position of the epitope of the antibody SKAI-48 of the present invention, and Figure 7c shows the three-dimensional structure of the epitope sequence of the antibody SKAI-48 of the present invention.

[0034] Figure 8 shows the results of FACS analysis to evaluate whether the anti-CT83 antibody-drug conjugate of the present invention specifically binds to cancer cells expressing the CT83 (KK-LC-1) antigen. The red histogram (left graph) represents the group treated with the anti-CT83 ADC of the present invention, and the blue histogram (right graph) represents the control group. In MB231 (breast cancer), HeLa (cervical cancer), H358 (lung cancer), KATOⅢ (gastric cancer), and MB468 (breast cancer) cells, a rightward shift in the fluorescence signal was observed upon treatment with the anti-CT83 ADC, confirming specific binding to the CT83 antigen. On the other hand, no signal change was observed in H1299 (lung cancer) and HEK293T (kidney-derived cells), indicating that the anti-CT83 ADC of the present invention does not cause non-specific binding in CT83-non-expressing cells.

[0035] Figure 9 shows the change in cell viability in CT83-expressing cancer cells when the anti-CT83 antibody-drug conjugate of the present invention is treated at various concentrations. Figure 9a shows the results of the cytotoxicity evaluation in MB231 (breast cancer) cells, Figure 9b shows the results in MB468 (breast cancer) cells, Figure 9c shows the results in HeLa (cervical cancer) cells, and Figure 9d shows the results in KATOⅢ (gastric cancer) cells.

[0036] Figure 10 shows the results of an in vivo animal experiment in which the anti-CT83 antibody-drug conjugate of the present invention was administered to cancer cell lines. Figure 10a shows the changes in tumor volume and survival effects over time following the administration of the anti-CT83 ADC in an animal model transplanted with CT83-positive cancer cell lines, and Figure 10b shows the changes in body weight and the occurrence of toxicity in the anti-CT83 ADC administration group and the control group.

[0037]

[0038] The present invention will be explained in more detail below with specific examples. However, the embodiments described below are provided as examples to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0039] Accordingly, the present invention is not limited to the embodiments presented below and may be embodied in other forms, and the embodiments presented below are described merely to clarify the concept of the present invention and are not limited thereto.

[0040]

[0041] definition

[0042] Expressions such as “comprising,” “comprising,” “having,” etc. as used herein should be understood as open-ended terms implying the possibility of including other embodiments in a manner similar to “comprising,” unless otherwise stated in the phrase or sentence containing such expressions.

[0043] The term "and / or" as used herein may mean any one or more of the items, any combination of the items, or all of the items in relation to the term.

[0044]

[0045] The present invention relates to an antibody-drug conjugate (ADC) in which a cytotoxic drug is conjugated to a CT83-specific antibody or an antigen-binding fragment thereof. The ADC of the present invention is designed to selectively bind to CT83-positive cancer cells in the body, and to selectively kill tumor cells by efficiently delivering the drug into the cell after binding. To this end, the antibody or antigen-binding fragment used in the present invention includes heavy chain and light chain variable regions that recognize the structural epitope of CT83 with high specificity and affinity, and said antibody can provide CT83 target selectivity as a key component constituting the ADC of the present invention.

[0046] The amino acid sequences of the antibody or antigen-binding fragments constituting the ADC of the present invention are shown in Table 2. The CT83 antigen is a protein expressed by the CT83 gene and is also known as CXorf61, KKLC1, Kita-kyushu lung cancer antigen 1, KK-LC-1, or Cancer / Testis antigen 83; it is known as one of the cancer-testis antigens having a molecular weight of approximately 12.8 kDa. Although CT83 is rarely expressed in normal tissues, it is selectively expressed in various solid tumors, making it suitable as a target antigen for antibody therapeutics and antibody-drug conjugates (ADCs).

[0047] As one embodiment for achieving the above technical problem, the antibody or antigen-binding fragment of the present invention may include a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO. 7, the heavy chain CDR2 of SEQ ID NO. 8, and the heavy chain CDR3 of SEQ ID NO. 9, and a light chain variable region comprising the light chain CDR1 of SEQ ID NO. 10, the light chain CDR2 of SEQ ID NO. 11, and the light chain CDR3 of SEQ ID NO. 12, and the antibody or the antigen-binding fragment thereof may specifically bind to the epitopes of SEQ ID NOs. 17 and 18.

[0048] An epitope refers to a specific region of an antigen that an antibody recognizes and binds to. Epitopes can be broadly classified into linear epitopes and conformational epitopes. Linear epitopes consist of a continuous sequence of amino acids and are primarily found in denatured proteins. In contrast, conformational epitopes are formed by the three-dimensional structure of a protein, occurring when amino acids that were previously separated become spatially closer due to protein folding. Most B cell epitopes (approximately 90%) are conformational; since conformational epitopes depend on the protein's inherent three-dimensional structure, denaturation destroys this structure, causing the protein to lose its binding affinity with antibodies.

[0049] Meanwhile, it can be confirmed that the epitope recognized by the antibody of the present invention or its antigen-binding fragment is a structural epitope as shown in FIG. 7c, and it can be seen that the SKAI-48 antibody binds dependently to the three-dimensional structure of the CT83 protein.

[0050] The phenomenon in which an antibody binds to two different epitopes simultaneously is known as "dual-Fab cis binding" or "bivalent binding." This means that two Fab portions of an antibody bind to different sites on the same antigen molecule. This binding mode can have a significant impact on the specificity and function of the antibody. Specifically, binding to two epitopes simultaneously can increase the binding strength and specificity of the antibody. In the case of the epitope of the present invention, since it corresponds to a structural epitope, the two sequences can be located close to each other in a three-dimensional structure, allowing the antibody to bind to two different epitopes simultaneously.

[0051] The antibody or its antigen-binding fragment of the present invention may be implemented in the form of a scFv. The scFv is a fragment antibody in which a heavy chain variable region and a light chain variable region are connected by a single chain, and because it has a relatively small molecular weight, it has the advantage of excellent tissue penetration and the ability to efficiently access the interior of a solid tumor.

[0052] The present invention provides an antibody-drug conjugate (ADC) in which an anticancer pharmaceutical substance is conjugated to the antibody or its antigen-binding fragment. The antibody-drug conjugate of the present invention selectively binds to and is internalized by cancer cells expressing CT83, and can induce cancer cell death by releasing a cytotoxic substance through a linker cleavage or drug release mechanism after internalization.

[0053] The drugs used in the present invention may include one or more selected from the group consisting of toxins, mitotic inhibitors, apoptosis inducers, anticancer chemotherapy agents, and antitumor agents; more specifically, they may include one or more selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), meitansinoid (DM1), caliceamicin, duokamycin, and anthracyclines. Most preferably, monomethylauristatin E (MMAE) may be used.

[0054] Meanwhile, the above antibody-drug conjugate may mean that a drug is conjugated to the antibody or its antigen-binding fragment through a linker.

[0055] In the present invention, the linker used to conjugate a drug to an antibody or its antigen-binding fragment may be any of the various known linkers designed to be selectively cleaved according to the environment inside the cancer cell or enzymatic action. Such linkers may include disulfide-bond-based linkers that are cleaved in a redox environment, peptide-based linkers that are cleaved by intracellular proteolytic enzymes, site-specific linkers designed to target only specific sites on the antibody, or autolytic linkers that self-degrade during the drug release process. Examples include valine-citrulline (Val-Cit) dipeptide linkers, glycine-phenylalanine-leucine-glycine (GFLG) peptide linkers, or maleimide-based linkers that react with and bind to the cysteine ​​group of the antibody.

[0056] Such a linker can operate to release a drug into the cell through a continuous autolysis process after being cleaved by proteolytic enzymes inside the tumor cell or by reducing conditions of the tumor microenvironment. Preferably, in the present invention, a linker comprising a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl structure such as Formula 1 may be used. The linker has the characteristic of being selectively cleaved inside the cancer cell to release the drug.

[0057] <Chemical Formula 1>

[0058]

[0059] The above-mentioned drug may be conjugated to an antibody or its antigen-binding fragment through a binding method with amino acid residues of the antibody, such as by reacting with and binding to the cysteine ​​group of the antibody or by site-specific binding performed by genetically designing a specific site of the antibody. Such binding methods can play an important role in controlling the stability of the antibody-drug conjugate in the blood, its half-life in vivo, and the selective drug release characteristics in the tumor microenvironment.

[0060] Furthermore, the antibody-drug conjugate according to the present invention can be effectively applied to various solid tumors expressing the CT83 (KK-LC-1) protein, such as cervical cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, bladder cancer, gastric cancer, and esophageal cancer, and can also be utilized as a therapeutic agent in some hematological cancers where CT83 expression is confirmed. Therefore, the antibody-drug conjugate of the present invention has high potential for application in various types of cancer as an anticancer treatment technology targeting CT83, and can serve as a candidate therapeutic agent that simultaneously satisfies tumor selectivity and drug release characteristics.

[0061] Specifically, the present invention may provide a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate as an active ingredient. The composition may be formulated with a pharmaceutically acceptable carrier and may be administered in various ways, such as intravenous or subcutaneous administration.

[0062] Furthermore, the present invention may provide a method for preventing or treating cancer expressing the CT83 (KK-LC-1) antigen. Specifically, by administering an effective amount of the antibody-drug conjugate according to the present invention to a subject, it selectively binds to cancer cells expressing the CT83 antigen to induce cytotoxicity, thereby enabling inhibition of tumor growth, reduction of tumor size, and inhibition of cancer progression. The method may be performed as a monotherapy, as well as in combination with radiation therapy, chemotherapy, or other anticancer treatments.

[0063] In addition, the present invention may provide for the use of the antibody-drug conjugate according to the present invention for the prevention or treatment of cancer expressing the CT83 (KK-LC-1) antigen. That is, the antibody-drug conjugate of the present invention may be used in the manufacture of a pharmaceutical composition for the prevention or treatment of cancer expressing the CT83 antigen, and may be utilized as an anticancer treatment technology that simultaneously satisfies tumor selectivity and drug release characteristics.

[0064] The cancers subject to the prevention or treatment mentioned above are cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple It may include multiple myeloid blood cancer and / or solid tumors.

[0065] Prevention or treatment of such cancer can be achieved by adjusting the appropriate route of administration, dosage, and administration interval according to the patient's condition, the type of cancer, and the stage of progression.

[0066]

[0067] The present invention will be explained in more detail below through the following examples. However, the examples are merely illustrative to easily explain the content and scope of the technical concept of the present invention, and the technical scope of the present invention is not limited or altered by these examples. Furthermore, it can be determined by those skilled in the art that various modifications and changes are possible within the scope of the technical concept of the present invention based on these examples.

[0068]

[0069] Example 1. Preparation of Anti-CT83 ADC

[0070] Example 1-1. Library Construction and Expression

[0071] To identify peptide sequences capable of specifically binding to CT83 protein, an antibody library was constructed and expressed in the following manner.

[0072] Biopanning for discovering antibody candidate sequences is 7.6 x 10 9 We used an OPAL library (OPAL library, oriented peptide array library, Construction of a Large synthetic human scFv library with six diversified CDRs and high functional diversity., Yang Hym at. al., Molecules and cells, 2009. DOI / 10.1007 / s10059-009-0028-9) in which an HA tag is attached to nucleotides encoding scFvs.

[0073] The antibodies expressed by the above OPAL library include a human variable heavy chain encoded by the gene Vh3-23 located at V3-23 and a human variable light chain encoded by the gene Vλ1g located at locus 1g, and CDR-H1, H2, L1, L2, and L3 include some CDR loops and CDR sequences combined or combined with human-derived CDR sequences.

[0074] CDR-H3 consists of four sublibraries, including AE, BE, CF, and DF (Table 1), and forms intraloop disulfide bonds (C-(X)4-C) between the cysteines of the DF sublibrary.

[0075]

[0076] CDR-H3AE(N-ter)-(RK)-(X)4-FDY-(C-ter)Sequence No. 1BE(N-ter)-(RK)-(DG)-(LPRVAG)-(X)7-FDY-(C-ter)Sequence No. 2CF(N-ter)-(RK)-(FSYCLPHRVADG)-(LPRITSVAG)-(X)7-(YS)-(YS)-(ADSY)-(YND)-(GA)-MDV-(C-ter)Sequence No. 3DF(N-ter)-(RK)-(VADG)-(LPRITSVAG)-(LRISVG)-XC-(X)4-C-(YS)-(YS)(ADSY)-(YND)-(GA)-MDV-(C-ter)Sequence No. 4

[0077]

[0078] X represents an arbitrary amino acid

[0079] In (X)n, n means that the corresponding amino acid is repeated n times.

[0080] The above OPAL library was transformed into ER2738 (E. Coli, AMID BIOSCIENCES, cat. No. ER-201), and 20 MOI of helper phage (VCSM13) was added to 2xYT medium (BD Difco™, 2xYT powder) containing 50 µg / ml carbenicillin and 70 µg / ml kanamycin, and cultured at 30°C for 14 hours. Subsequently, the bacteriophage was activated (rescue) using 4% Polyethylene Glycol 8000 (PEG 8000).

[0081]

[0082] Example 1-2. Selection of candidate proteins that specifically bind to CT83 protein

[0083] Biopanning was performed as follows to select candidates that specifically bind to the CT83 protein.

[0084] Specifically, biopanning was performed using recombinant human lung cancer antigen 1 (CT83)-VLPs (KK-LC-1 VLP, CSB-MP711093HU, CUSABIO) (Table 2) to search for substances that specifically bind to the (CT83)-VLPs antigen.

[0085]

[0086] Protein Sequence Number CAT#Amino Acid Sequence Expression Cell Tag KK-LC-1(CT83)(NM_001017978)5TP309391(N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter)HEK293T(C-ter)-Myc / DDK Recombinant Human Lung Cancer Antigen 1(CT83)-VLPs(active)6CSB-MP711093HU(N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter)Mammalian cells(N-ter)- 6xHis-tag

[0087]

[0088] The CT83 antigen was conjugated to epoxy magnet beads (M-270 Epoxy) at 4°C overnight, and then incubated with scFv-expressing phages at 37°C for 2 hours. Subsequently, the CT83-conjugated phages were eluted using 100 mM Triethylamine (TEA) and then infected with new ER2738 cells. The biopanning process, in which the scFv-expressing bacteriophages were specifically conjugated to the CT83 antigen and then eluted using TEA, was repeated three times. By repeating the biopanning process, substances that bind non-specifically to the antigen could be removed.

[0089] Subsequently, to filter out substances that bind non-specifically to the VLP itself, a biopanning was performed one more time using KK-LC-1 (CT83) (NM_001017978) Human Recombinant Protein (CAT#: TP309391, Origene) (Table 2) to discover antibody candidates.

[0090] 400 monoclonal candidate antibodies were randomly selected from colonies that formed independently among the colonies cultured after the four pannings mentioned above (Fig. 1). ELISA analysis was performed on each candidate antibody expressed in the scFv form, and 50 antibody candidate antibodies that specifically bind to KK-LC-1 (CT-83) relative to BSA and KK-LC-1 VLPs relative to BSA were selected.

[0091]

[0092] Examples 1-3. NGS analysis of selected candidates and selection of final candidates

[0093] For 50 selected candidates that bind to KK-LC-1 (CT83) and KK-LC-1 VLPs but do not bind to BSA and VLP-(negative), the gene sequences of each of the 50 candidates were analyzed through Next Generation Sequencing (NGS) gene analysis.

[0094] NGS analysis was performed on the 50 selected antibody candidates to exclude duplicate antibodies and antibodies with frame shifts, and 46 antibody candidates were selected. The scFvs of the 46 antibody candidates were reproduced using gene sequences, and the recombinant KK-LC-1 / BSA and KK-LC-1 VLPs / BSA were analyzed by ELISA to select the final antibody candidates capable of specifically binding to CT83 protein.

[0095] As a result, four final antibody candidates were selected in which the absorbance ratio exceeded 10 and the absorbance of the negative control was below the threshold. The final antibody candidates were named SKAI-5, SKAI-15, SKAI-33, and SKAI-48, and the heavy chain and light chain CDR amino acid sequences of SKAI-48 among the antibody candidates are listed in Table 3.

[0096] The ELISA results of SKAI-48 are shown in Figure 2. After adding TMB (3,3',5,5'-tetramethylbenzidine) and 20 minutes later, the absorbance was measured at 450 nm using a plate reader.

[0097]

[0098] SKAI-48VHCDR1CDR2CDR3 Sequence Number 7 Sequence Number 8 Sequence Number 9(N-ter)-DYYMS-(C-ter)(N-ter)-AISYDSSST-(C-ter)(N-ter)-ARKKKKFDY-(C-ter)VLCDR1CDR2CDR3 Sequence Number 10 Sequence Number 11 Sequence Number 12(N-ter)-SGSSSNIGSNNVT-(C-ter)(N-ter)-ANSN-(C-ter)(N-ter)-GTWDASLSG-(C-ter)

[0099]

[0100] Examples 1-4. Identification of epitope sequences binding to CT83

[0101] The final antibody candidate identified in Example 3 was prepared as an IgG antibody, and the following experiment was performed to clearly identify the amino acid sequence of the binding site of the antibody to the CT83 protein.

[0102] First, the scFv sequence of SKAI-48 binding to CT83 was inserted into a human IgG two-vector system (TGEX-HC, Antibody design labs, MX001, and TGEX-LC, Antibody design labs, MX002) in the following manner.

[0103] Specifically, a gene cloning vector was constructed to amplify the antibody gene binding site using forward and reverse primers containing restriction enzyme sites, thereby enabling the production of IgG using a nucleotide sequence encoding scFv. The nucleotide sequences of the primers are listed in Table 4.

[0104]

[0105] Vector Name Forward Primer (5'->3') Reverse Primer (5'->3') TGEX-VH Sequence No. 13 Sequence No. 14 (BSSHll)NNGCGCGCACTCCGAGGTGCAGCTGTTG(BsmBl)NNCGTCTCNATGCTGAGCTCACGGTGACTGEX-VL Sequence No. 15 Sequence No. 16 (BspEI)NNNNNNTCCGGACAGTCTGTGCTGACT(BsaI)NNGGTCTCNTTCGTAGGACCGTCAGCT

[0106]

[0107] A vector was constructed in the following manner to enable the expression of the above IgG in mammalian cells.

[0108] The VH primer was designed to have restriction sites for BSSHll at the 5' end and BsmBl at the 3' end, and the VL primer was designed to have restriction sites for BspEI at the 5' end and BsaI at the 3' end. The nucleotide sequences of the VH primer are the nucleotide sequences of SEQ ID NOs. 13 and 14, and the nucleotide sequences of the VL primer are the nucleotide sequences of SEQ ID NOs. 15 and 16.

[0109] The amplified VH fragment was inserted into the TGEX-HC (Antibody design labs, MX001) vector, and the VL fragment was inserted into the TGEX-LC (Antibody design labs, MX002) vector. The plasmids were amplified using Escherichia coli DH5α-competent cells, and cloning was confirmed by DNA sequencing.

[0110] Cloned heavy and light chain human IgG plasmids were co-transfected into Expi-CHO-s cells in the following manner.

[0111] 6x10 6Expi-CHO-s cells were prepared, and 15 µg each of heavy and light chain IgG plasmids were incubated in Opti-pro SFM medium (ThermoFisher scientific, Cat no. 12309019) for 5 minutes. After 5 minutes, a complex was formed by mixing with Expifectamine™ CHO. The complex was transfected into the prepared Expi-CHO-s cells (ThermoFisher scientific, A29127) without exceeding the 5-minute reaction time. The next day, ExpiCHO™ Feed and ExpiFectamine™ CHO Enhancer (ExpiFectamine™ CHO Transfection kit, ThermoFisher scientific, Cat no. A29129), which provide nutrients and enhance protein production efficiency, were added. Cells were harvested when the cell viability reached approximately 75% (about 7 days after transfection).

[0112] To purify the generated antibody, the monoclonal antibody was purified using Protein A Resin (Amicogen). The generated antibody (volume 50 ml) and 1 ml of Protein A Resin (≥ 40 mg human IgG / ml resin) were mixed and incubated at 4°C for 6 hours. The mixture was then placed in an affinity chromatography column and eluted using 0.1 M glycine HCl buffer (pH 2.7). Finally, the purified antibody was concentrated using a 50 kDa cut-off amicon (Merck Millipore), and the antibody concentration was measured using a protein analysis kit (Pierce™, BCA Protein Assay kit, Thermo Scientific).

[0113] Afterwards, epitope mapping was performed using a microarray to identify the antigen binding sites (epitopes) of the selected antibodies against the target antigen (CT83).

[0114] Specifically, the SKAI-48 antibody prepared according to Example 4-1 was biotinylated before performing the microarray. Subsequently, the biotinylated antibody sample was analyzed on approximately 18,000 CT83 antigen peptide sites to confirm binding with the CT83 antigen (Fig. 3).

[0115] Antigen-antibody binding was confirmed by measuring the duplicated biotinylation signal. Information on the sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the major epitope binding to SKAI-48 is shown in Figures 6a and 6b, and the position of the repeat sequence was confirmed (Figure 7). In particular, the three-dimensional structure of the epitope binding to SKAI-48 of the present invention is illustrated in Figure 7c.

[0116] The identified epitope sequences are listed in Table 5 below.

[0117]

[0118] SKAI-48 Epitope 1 (Sequence No. 17) Epitope 2 (Sequence No. 18)(N-ter)-LVELEHT-(C-ter)(N-ter)-FRGASPHRKST-(C-ter)

[0119]

[0120] The simulation method performed to obtain the three-dimensional geometry of the structure of CT83 in Fig. 7c is as follows. The CT83 protein sequence (NP_001017978.1) was obtained from the NCBI database (https: / www.ncbi.nlm.nih.gov / ). Since there were no identical or similar sequences in either the NCBI or PDB databases, the AlphaFold homologous model (AF-Q5H943-F1) provided by the EMBL AlphaFold server (https: / alphafold.ebi.ac.uk / ) was used for structural analysis. The quality of the model was evaluated through Ramachandran and ERRAT plots generated using the SAVES v6.0 web server (https: / saves.mbi.ucla.edu / ), and to further refine the model, a molecular dynamics (MD) simulation system of CT83 contained in the POPC lipid bilayer was prepared using the CHARMM-GUI web server (https: / www.charmm-gui.org / ). The POPC bilayer was selected because CT83 acts as a transmembrane domain and signal anchor for membrane proteins.

[0121] The epitope sequences of the SKAI-48 antibody were identified as SEQ ID NOs 17 and 18 above, and these sequences correspond to the parts that form a specific three-dimensional structure of the CT83 protein, rather than a simple linear peptide.

[0122] The fact that the epitope of the SKAI-48 antibody is a structural epitope has significant implications for the specificity and function of this antibody. Since structural epitopes depend on the unique three-dimensional structure of a protein, the SKAI-48 antibody can recognize specific structural features of the CT83 protein. This means that the SKAI-48 antibody can recognize the CT83 protein with high specificity, which can be a significant advantage when used for diagnostic and therapeutic purposes.

[0123]

[0124] Examples 1-5. Preparation of Anti-CT83 ADC

[0125] An anti-CT83 ADC was prepared using the SKAI-48 antibody identified in Examples 1-1 to 1-4 above and the cytotoxic drug monomethylauristatin E (MMAE). For drug conjugation, a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PABC) linker was used.

[0126] The above MC-Val-Cit-PABC linker contains a PABC (p-aminobenzyloxycarbonyl) motif having a protease-cleavable (valine-citrulline) structure and a self-immolative mechanism, and is designed to be covalently conjugated through the reduced cysteine ​​residue (Cys) and maleimide reaction group (MC) of the antibody. An anti-CT83 antibody-drug conjugate (anti-CT83 ADC) was prepared by reacting the MMAE-linker construct (MC-Val-Cit-PABC-MMAE) with the antibody following a partial reduction process of the SKAI-48 antibody.

[0127]

[0128] Experimental Example 1. Evaluation of CT83 Antigen Binding Specificity of Anti-CT83 ADC (FACS Analysis)

[0129] To determine whether the antibody-drug conjugate bound to the SKAI-48 antibody of the present invention specifically binds to the CT83(KK-LC-1) antigen expressed in cancer cells, a FACS experiment was conducted as follows. The MB231, HeLa, H358, KatoⅢ, and MB468 cells used in this experiment are cancer cells expressing the CT83(KK-LC-1) protein, while the H1299 and HEK293T cells are cancer cells not expressing the CT83(KK-LC-1) protein.

[0130] For the FACS experiment, each cell was collected by trypsin treatment, washed with PBS, and centrifuged to precipitate the cells, a process repeated twice. Subsequently, the anti-CT83 ADC solution was diluted to 10 µg / ml and incubated on ice for 30 minutes. Then, Allophycocyanin-labeled goat anti-human IgG antibody (R&D systems) was added at a concentration of 1:50, and after another 30 minutes of incubation, the cells were analyzed using forward scattering light (FSC).

[0131] As a result of the FACS experiment, as shown in Figure 8, a rightward shift in the fluorescence signal was observed in the MB231, HeLa, H358, Kato III, and MB468 cells used in which CT83(KK-LC-1) protein was expressed upon treatment with the anti-CT83 ADC, whereas in the H1299 and HEK293T cells in which CT83(KK-LC-1) was not expressed, the signal distribution was almost identical to that of the control group, confirming that the anti-CT83 ADC of the present invention specifically binds to the CT83 antigen.

[0132]

[0133] Experimental Example 2. Octet-based KD Measurement Experiment of Anti-CT83 ADC

[0134] To confirm the binding affinity of the antibody-drug conjugate bound to the SKAI-48 antibody of the present invention for the CT83 (KK-LC-1) protein, Octet-based Bio-Layer Interferometry (BLI) analysis was performed as follows. In this experiment, the antibody portion (SKAI-48, Clone 2352) of the anti-CT83 ADC was immobilized on an AR2G biosensor, and then the antigen-antibody binding rate and dissociation rate were measured by treating with recombinant CT83 (KK-LC-1) protein at various concentrations.

[0135] For the experiment, the anti-CT83 antibody was prepared at a concentration of 10 µg / ml and immobilized on the surface of the AR2G biosensor in sodium acetate (pH 6.0) buffer for 600 seconds. Under these conditions, the degree of immobilization was confirmed to be approximately 2.5 nm. Subsequently, the antigen (recombinant Human KK-LC-1, CT83 protein) was diluted to concentrations of 25, 50, 100, and 200 nM in the same buffer and analyzed. At each concentration, the binding time was set to 900 seconds and the dissociation time to 3600 seconds, and all data were fitted using a 1:1 binding model.

[0136] As shown in Table 6 below, the results of the octet analysis revealed that the antibody portion of the anti-CT83 ADC exhibited a concentration-dependent increase in the binding signal with the CT83 (KK-LC-1) protein, and all derived kinetic values ​​satisfied the Quality Control criteria (coefficient of determination R² ≥ 0.95, Ka / Kd error ≤ 10%, residual ≤ 10% of the reaction signal). The analysis showed that the final equilibrium dissociation constant (KD) of the antibody portion (SKAI-48, Clone 2352) of the anti-CT83 ADC of the present invention was 2.780 × 10⁻⁶. -9 It was calculated as M, which means that the anti-CT83 ADC of the present invention has a high binding affinity at the nanomolar (nM) level for the CT83(KK-LC-1) antigen.

[0137] From the above results, it was confirmed that the anti-CT83 ADC of the present invention possesses high binding strength and selectivity in binding to the CT83(KK-LC-1) antigen, and that it can be very suitablely utilized as an antibody-drug conjugate platform targeting CT83-expressing cancer cells.

[0138]

[0139] Conc. (nM)ResponseKD (M)KD Errorka (1 / Ms)ka Errorkdis (1 / s)kdis Error1000.15982.780x10 -9 8.130x10 -12 1.200x10 4 5.805x103.336x10 -5 8.485x10 -8 500.0871250.050612.50.0309

[0140]

[0141] Experimental Example 3. Evaluation of Anti-CT83 ADC Efficacy by Tumor Type

[0142] To determine whether the anti-CT83 antibody-drug conjugate (SKAI-48 ADC) of the present invention induces apoptosis in cancer cells expressing the CT83(KK-LC-1) antigen, a cell-based (in vitro) cytotoxicity assay was performed as follows. All cell lines used in this experiment are cancer cell lines expressing CT83(KK-LC-1), and MB231 and MB468 cells are breast cancer cell lines, HeLa cells are cervical cancer cell lines, and KATOⅢ cells are gastric cancer cell lines.

[0143] Each cell line was seeded at a density of 5,000 cells / well in white 96-well culture plates at a volume of 100 μL. After incubating for a certain period to allow cell attachment, 100 μL / well of the anti-CT83 ADC (SKAI-48-MMAE) of the present invention, serially diluted in RPMI medium (containing 10% FBS), was added. In this analysis, the final concentration of the anti-CT83 ADC ranged from 10 μg / mL to 0.64 ng / mL. The treated plates were incubated for 72 hours at 37°C under 5% CO₂ conditions.

[0144] After 72 hours, Cell Counting Kit-8 (DOJINDO) was added to each well and cultured for an additional 2 hours; then, cell viability was calculated by measuring absorbance according to the manufacturer's instructions. Wells containing only cells were set as the 100% viability baseline, and viability by concentration was analyzed using a 4-parameter logistic curve with GraphPad Prism to determine the IC50. 50 The value was calculated.

[0145] As a result, as shown in Table 7 and Figure 9 below, the anti-CT83 ADC of the present invention (SKAI-48) exhibited concentration-dependent cytotoxicity in all cancer cell lines expressing CT83 (KK-LC-1). In particular, in MB468 breast cancer cells, IC 50 The value was found to be very low at 5.5 ng / mL, confirming that the anti-CT83 ADC exhibits a potent apoptotic effect. Additionally, a low IC50 of 25.1 ng / mL was observed in the gastric cancer cell line KATO III. 50 The values ​​were confirmed to be 102.4 ng / mL in the cervical cancer cell line HeLa and 115.3 ng / mL in the breast cancer cell line MB231. 50 The values ​​were derived for each.

[0146] From these results, it was confirmed that the anti-CT83 ADC (SKAI-48) of the present invention can induce selective and potent cytotoxicity in cancer cells by targeting CT83 (KK-LC-1). This suggests that the ADC of the present invention can be usefully utilized as a therapeutic candidate for CT83-expressing cancers.

[0147]

[0148] Cellular carcinoma IC 50 (ng / ml) MB231 Breast cancer 326.1 MB468 Breast cancer 84.2 HeLa Cervical cancer 181.7 KATOⅢ Gastric cancer 77.9

[0149]

[0150] Experimental Example 4. Evaluation of In Vivo Tumor Suppressive Efficacy of Anti-CT83 ADC

[0151] To confirm whether the anti-CT83 antibody-drug conjugate of the present invention (hereinafter “anti-CT83 ADC”) exhibits antitumor activity in carcinomas expressing CT83 (KK-LC-1), an in vivo evaluation of antitumor efficacy was performed in a human xenograft mouse model. In this example, a subcutaneous (sc) xenograft model using MDA-MB-231 cells, a breast cancer cell line expressing CT83, was used.

[0152] 5×10⁶ MDA-MB-231 cells (CT83 positive) 6 Tumors were formed by injecting the anti-CT83 ADC into the right flank of BALB / c nude female mice one by one. When the tumor size reached approximately 100-150 mm³, the mice were randomly reclassified into five groups. The anti-CT83 ADC was used for the test group, and the controls were (i) unconjugated anti-CT83 monoclonal antibody (mAb), (ii) unrelated antigen-specific IgG (homogeneous control), and (iii) PBS, respectively. The anti-CT83 ADC was administered via tail vein (IV) at a dose of 3 mg / kg for a total of three times on days 0, 7, and 14.

[0153] After treatment, the tumor size was measured twice a week using a caliper to determine length and width, and the tumor volume (mm³) was calculated using the formula 0.52 × (length) × (width)². In addition, safety indicators were monitored in parallel through changes in mouse body weight, activity, and gross organ findings.

[0154] As a result, as shown in Fig. 10, it was confirmed that the anti-CT83 ADC (#48-ADC) prepared according to the embodiment of the present invention reacts with the CT83 antigen and exhibits a toxic effect on cancer cell lines. Specifically, as shown in Fig. 10a, in an animal model experiment using cancer cell lines, the group administered the anti-CT83 ADC prepared according to the embodiment of the present invention showed a decrease in tumor volume and an extension of survival time compared to the control group. Furthermore, as shown in Fig. 10b, there were no deaths due to toxicity in the group administered the anti-CT83 ADC (#48-ADC), and body weight was similar in both the control and treatment groups. Through this, it was found that the anti-CT83 ADC prepared according to the embodiment of the present invention has excellent anticancer efficacy in vivo.

[0155]

[0156] As such, the present invention relates to an antibody-drug conjugate (ADC) based on the novel antibody SKAI-48, which targets the CT83 (KK-LC-1) antigen, wherein the antibody-drug conjugate exhibits selective binding and cytotoxicity against CT83-expressing cancer cells. Through FACS analysis, it was confirmed that the anti-CT83 ADC of the present invention can selectively target the CT83 antigen, and through in vitro cytotoxicity analysis, it was confirmed that the ADC of the present invention can exhibit a concentration-dependent effect of reducing survival in cancer cells with high CT83 expression.

[0157] The anti-CT83 ADC of the present invention utilizes the tumor-selective expression characteristics of the CT83 antigen, thereby minimizing non-specific toxicity in normal tissues and selectively delivering the drug to tumor tissues. This can reduce the problem of systemic toxicity, which was a limitation of existing anticancer drugs, and can demonstrate therapeutic effects against various cancers expressing CT83. High binding affinity with nanomolar-level KD values ​​enables strong selectivity for tumor tissues, which can provide clinical advantages in terms of therapeutic efficacy and safety.

[0158] In addition, the anti-CT83 ADC of the present invention can be expanded into a next-generation anticancer platform by combining with various drugs. For example, by combining with MMAE, DNA damage inducers, radioisotopes, etc., a customized anticancer treatment strategy tailored to tumor characteristics can be implemented. This platform-based expandability implies that it can be utilized as a precision medicine-based therapeutic agent for CT83-positive patients through future preclinical and clinical development.

[0159] In summary, the present invention provides an antibody that highly specifically recognizes CT83 and an ADC technology based thereon, thereby solving the problems of existing technologies such as tumor-selective cytotoxicity induction, high binding affinity, reduced toxicity to normal tissues, and platform scalability, and can have high industrial and clinical potential in the field of anticancer drug development.

[0160] Embodiments of the present invention have been described above. The technical features are not limited thereto and include all variations that can be modified and applied by a person skilled in the art based on the technical concept of the present invention.

Claims

As an antibody-drug conjugate in which a drug is conjugated to an antibody that specifically binds to CT83 or its antigen-binding fragment, An antibody-drug conjugate characterized in that the antibody or its antigen-binding fragment comprises a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO. 7, the heavy chain CDR2 of SEQ ID NO. 8, and the heavy chain CDR3 of SEQ ID NO. 9, and a light chain variable region including the light chain CDR1 of SEQ ID NO. 10, the light chain CDR2 of SEQ ID NO. 11, and the light chain CDR3 of SEQ ID NO.

12. In paragraph 1, An antibody-drug conjugate characterized in that the antibody or its antigen-binding fragment specifically binds to the epitopes of SEQ ID NOs 17 and 18. In paragraph 1, An antibody-drug conjugate characterized in that the antibody or its antigen-binding fragment is a scFv. In paragraph 1, The above-mentioned drug is an antibody-drug conjugate characterized by comprising one or more selected from the group consisting of toxins, mitotic inhibitors, apoptosis inducers, anticancer chemotherapy agents, and antitumor agents. In paragraph 1, The above-mentioned drug is an antibody-drug conjugate characterized by comprising one or more selected from the group consisting of monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), meitansinoid (DM1), caliceamicin, duocamycin, and anthracycline. In paragraph 1, An antibody-drug conjugate characterized in that the above drug is monomethylauristatin E (MMAE). In paragraph 1, The above antibody or its antigen-binding fragment and the drug are conjugated through a linker, and An antibody-drug conjugate characterized in that the above linker has the structure of Chemical Formula 1. <Chemical Formula 1> A pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate of claim 1 as an active ingredient. In paragraph 8, The above cancers include cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain tumor, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple A pharmaceutical composition for the prevention or treatment of cancer, characterized by comprising one or more selected from the group consisting of multiple myeloid blood cancers and solid tumors. A method for the prevention or treatment of cancer comprising administering an effective amount of the antibody-drug conjugate of claim 1 to a cancer patient. In Paragraph 10, The above cancers include cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain tumor, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple A method for preventing or treating cancer, characterized by comprising one or more selected from the group consisting of multiple myeloid blood cancers and solid tumors. Use of the antibody-drug conjugate of claim 1 for the prevention or treatment of cancer.

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