KR102572039B1 - c-Kit targeting Immunoconjugate comprising Pseudomonas exotoxin A - Google Patents

Abstract

The present invention relates to an immunoconjugate comprising Pseudomonas exotoxin A (PE), and specifically, an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to a specific region of c-Kit, photoreactive The present invention relates to an immunoconjugate comprising an Fc regioselective binding peptide containing an amino acid and Pseudomonas exotoxin A, and a pharmaceutical composition for preventing or treating c-Kit-positive cancer comprising the immunoconjugate as an active ingredient. The immunoconjugate of the present invention exhibits excellent anticancer activity by specifically binding to a specific region of c-Kit, inhibiting intracellular protein synthesis, and exhibiting cytotoxicity against c-Kit-positive cells.

Description

c-Kit targeting immunoconjugate comprising Pseudomonas exotoxin A {c-Kit targeting Immunoconjugate comprising Pseudomonas exotoxin A}

The present invention relates to a c-Kit targeting immunoconjugate comprising Pseudomonas exotoxin A (PE), and specifically, an anti-c-Kit antibody that specifically binds to a specific region of c-Kit or an antigen binding thereof. The present invention relates to an immunoconjugate comprising a fragment, an Fc regioselective binding peptide comprising a photoreactive amino acid, and Pseudomonas exotoxin A, and a pharmaceutical composition for preventing or treating c-Kit-positive cancer comprising the immunoconjugate as an active ingredient. .

An antibody-drug conjugate consists of three components: an antibody targeting a cell surface antigen expressed by cancer cells, a cytotoxic payload, and a linker attaching the payload to the antibody. ADC minimizes the negative effect of the payload on normal tissues and is internalized into lysosomes by forming complexes with target antigens. The cytotoxic payload is then released by proteolytic enzymes and enters the cytoplasm, leading to cytotoxicity and cancer cell apoptosis.

The selection of cytotoxic payloads is critical for successful treatment of patients with various tumor types. Currently, payloads with various mechanisms of action, including DNA alkylating agents, RNA polymerase inhibitors, DNA topoisomerase inhibitors, microtubule inhibitors, and RNA splicing inhibitors, are being used for ADC development. Inappropriate payload selection may result in lack of efficacy or serious side effects in cancer patients, so factors such as mechanism of action, efficacy, bystander effect, and multidrug resistance should be considered when selecting a payload. ADCs including Mylotarg, Adcetris, Cardcilla, Vesponsa and Enhertu have been approved for the treatment of cancer patients, and more than 150 ADCs are currently in clinical trials.

Meanwhile, stem cell factor (SCF) plays an important role in hematopoietic and reproductive cell development as well as stimulating the development and regulating the functions of mast cells and melanocytes. SCF can exist as an 18-kDa soluble form or as a 31-kDa membrane-bound molecule that differs in exon 6 of the transmembrane segment. The SCF receptor, c-Kit, is a class III subfamily that includes platelet-derived growth factor (PDGFR), macrophage colony-stimulating factor 1, and FMS-like TK-3/embryonic liver kinase-2 (FLT-3/FLT-2) receptors. of the tyrosine kinase receptor. Upon SCF binding, c-Kit is activated by phosphorylation of several tyrosine residues, leading to cell proliferation, survival, differentiation and chemotaxis. The SCF/c-Kit signal is subject to negative feedback control. It is downregulated by intracellular uptake of the receptor at the cell surface, clathrin-mediated internalization, ubiquitination and proteasome-mediated degradation. Under normal circumstances, c-Kit activation is tightly regulated and is associated with the development of malignancies such as acute myeloid leukemia (AML), gastrointestinal stromal tumors (GIST), melanoma and mast cell tumors.

Previous studies have reported that ADCs containing DM1 efficiently inhibit tumor growth in mouse xenograft models transplanted with c-Kit-positive small cell lung cancer and leukemia (Mol Oncol 16(6): 1290-1308, 2022 ). However, some tumors did not respond efficiently to c-Kit-targeted ADCs.

Under this background, the present inventors have made diligent efforts to develop a novel therapeutic agent that can effectively treat c-Kit-positive cancer, and as a result, confirmed that the immunoconjugate of the present invention has an excellent anti-cancer effect in c-Kit-positive cancer, and the present invention has been completed.

Mol Oncol 16(6): 1290-1308, 2022

One object of the present invention is to provide an immunoconjugate comprising an anti-c-Kit antibody or antigen-binding fragment thereof, an Fc regioselective binding peptide comprising a photoreactive amino acid, and Pseudomonas exotoxin A, or a pharmaceutically acceptable salt thereof. is to do

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating c-Kit-positive cancer comprising the immunoconjugate or a pharmaceutically acceptable salt thereof.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

I. Immunoconjugates

One aspect of the present invention for achieving the above object is an immunoconjugate represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

Ab-(L-D)p

In the above formula,

Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to sites D113 to P206 of SEQ ID NO: 20 in c-Kit;

L is an Fc regioselective binding peptide containing a photoreactive amino acid;

D is Pseudomonas exotoxin A; and

p is an integer from 1 to 10;

In the present invention, the term "immunoconjugate" refers to a complex in which an antibody or an antigen-binding fragment thereof is linked to a drug-linker conjugate. In the present invention, the term "drug-linker conjugate" refers to a material for preparing an immunoconjugate, in which an antibody or an antigen-binding fragment thereof is not linked, and binds to any antibody or antigen-binding fragment thereof as desired to immunize the body. Can be used as a conjugate. When the immunoconjugate is administered in vivo, the antibody or antigen-binding fragment thereof binds to the target antigen and then releases the drug to allow the drug to act on target cells and/or surrounding cells, resulting in excellent efficacy and reduced drug efficacy as a target drug. Side effects can be expected. In particular, the immunoconjugate of the present invention is an antibody or antigen-binding fragment thereof that binds to a specific region of c-Kit, an Fc-binding peptide containing a photoreactive amino acid, and Pseudomonas exotoxin A means combined.

In the present invention, the term "antibody" refers to a protein molecule that serves as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, Includes all whole antibodies. The term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds that have a binding function to FcRn. A full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

As used herein, the term "heavy chain" refers to a full-length heavy chain including a heavy chain variable region and a heavy chain constant region and fragments thereof. There are gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types of heavy chains.

As used herein, the term "light chain" refers to a full-length light chain including a light chain variable region and a light chain constant region, and fragments thereof. There are kappa (κ) and lambda (λ) types of light chains.

In the present invention, the antibody is a full-length antibody or an antibody fragment having antigen-binding ability, and the heavy chain may be any one of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type, Light chains can be of the kappa (κ) or lambda (λ) type.

The term "c-Kit" used in the present invention belongs to class III of Receptor Tyrosine Kinase (RTK) and is also known as the SCF receptor. Upon ligand binding, c-Kit is activated by autophosphorylation via ligand-mediated dimerization. Consequently, signaling mediators including Src, PI3K, STAT1 and JAK2 are recruited to regulate various cell-specific responses such as proliferation, survival and motility. In addition, overexpression of RTK is known to activate spontaneously to induce cell growth in a ligand-independent manner. Overexpression and activation of c-Kit have been reported in a variety of cancers, including GBM, astrocytoma, germ cell and SCLC, and are correlated with poor prognosis.

In the present invention, the term "anti-c-Kit antibody" refers to an antibody that specifically binds to c-Kit, and in particular, the anti-c-Kit antibody of the present invention is domain II of c-Kit, specifically SEQ ID NO: 20 It binds specifically to specific epitopes from D113 to P206 and thereby inhibits or neutralizes the activity or activation of c-Kit. Here, the sequence set forth in SEQ ID NO: 20 has meaning as a reference sequence for specifying an epitope, and if the antibody binds to the same epitope as the antibody of the present invention, the sequence of c-Kit to be targeted is SEQ ID NO: 20 and the like by mutation or the like Even if they are partially different, they may all be included in the scope of the present invention. In addition, even when some sequences from D113 to P206 of SEQ ID NO: 20 are added, substituted, or deleted, it should be understood that all are included in the scope of the present invention as long as they have activity similar to that of the antibody of the present invention.

In one example, the antibody may include, but is not limited to, a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2, and a light chain CDR3 of SEQ ID NO: 3; And a reference antibody comprising 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 human c-Kit to bind competitively to each other. can In another example, the antibody comprises a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2, and a light chain CDR3 of SEQ ID NO: 3; and 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, even if some amino acids in the CDR are added, deleted or substituted. All are included in the scope of the present invention as long as they bind to an epitope. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

As another example of the present invention, the antibody may include a light chain variable region represented by SEQ ID NO: 4 and a heavy chain variable region represented by SEQ ID NO: 10.

In the present invention, the anti-c-Kit antibody may include a constant region derived from human IgG1 and may be a human anti-c-Kit antibody.

As used herein, the term "human antibody" can be understood to mean an antibody having variable regions in which the framework and/or CDR regions are derived from human immunoglobulin sequences. A human antibody in the present invention may comprise amino acid residues not encoded by human-derived immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). there is. The human antibody may be in the form of a full length antibody or a form comprising a functional fragment of an antibody molecule. Since all components of the human antibody are derived from humans, the probability of an immune response occurring when administered to a human is low compared to a humanized antibody or a mouse antibody. Therefore, it has advantageous advantages as a therapeutic antibody for humans.

As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, the contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope can be, for example, a polypeptide or two or more non-contiguous regions of polypeptides joined together (conformational epitope, non-linear, discontinuous, or non-contiguous epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, and epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, and overlapping or contiguous peptides (eg, c -Kit domains 2 and 3) are tested for reactivity with a given antibody (eg an anti-c-Kit antibody). Methods for determining the spatial conformation of an epitope include those skilled in the art and those disclosed herein, and include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (G. E. Morris, Ed., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, 1996).

The term "binds to the same epitope" in the context of two or more antibodies means that the antibodies bind to the same segment of amino acid residues as determined by a given method. Techniques for determining whether antibodies bind to the "same epitope on c-Kit" as the antibodies presented herein include, for example, epitope mapping methods, such as x-rays of crystals of antigen:antibody complexes that provide atomic resolution of the epitope. analysis and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Another method monitors the binding of an antibody to an antigen fragment or mutated variant of an antigen, where loss of binding due to alteration of an amino acid residue within the antigen sequence is considered primarily an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of an antibody of interest to affinity separate specific short peptides from combinatorial phage display peptide libraries. Antibodies with identical VH and VL or identical CDR sequences are expected to bind to the same epitope.

An antibody that "competitively binds to another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to the target. Whether or not two antibodies compete with each other for binding to the target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined using known competition experiments. In certain embodiments, an antibody competes for binding to a target with another antibody and reduces this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. hinder The level of inhibition or competition may differ depending on whether the antibody is a “blocking antibody” (ie, a cold antibody that has been first incubated with the target). Competitive analysis is, for example, E.d. Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.prot4277 or E.d. It can be performed as set forth in Chapter 11 of “Using Antibodies” by Harlow and David Lane (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999). Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (eg, by steric hindrance).

The term "reference antibody" refers to an antibody that is a reference for analysis of a competitor antibody that binds to the same epitope, overlapping epitope, or adjacent epitope, and cross-competes in binding with the competitor antibody and the epitope. )do.

Other competitive binding assays include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using the I-125 label (see Morel et al., Mol. Immunol . 25(1):7 (1988)); solid phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the term "antigen-binding fragment" refers to any fragment of an antibody of the present invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab') ₂ , dsFv or scFv. The Fd refers to the heavy chain part included in the Fab fragment. The Fab has a structure having light chain and heavy chain variable regions, light chain constant region, and heavy chain first constant region (CH1 domain), and has one antigen binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab') ₂ antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and light chain variable region are linked by a disulfide bond, and in single chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab') ₂ fragments can be obtained by digestion with pepsin), preferably Preferably, it can be produced through genetic recombination technology.

In the present invention, the Fc regioselective binding peptide (FcIII) refers to a peptide that regiospecifically binds to the CH ₃ -CH ₂ interface region of the Fc domain of a human antibody. The peptide exhibits a U-shaped structure because two cysteines form a disulfide bond with each other. The Fc regioselective binding peptides of the present invention are characterized by including "photoreactive amino acids".

In the present invention, "photoreactive amino acid" refers to a functional group capable of forming a covalent bond with an adjacent reactive functional group by absorbing light of a specific wavelength when irradiated with light. When the Fc regioselective binding peptide comprising the photoreactive amino acid of the present invention is mixed with an antibody, it is adjacent to or binds to the Fc domain of the antibody by its inherent specificity. Thereafter, when the mixture is irradiated with light, it can absorb light of a specific wavelength and form a covalent bond with a reactive functional group on the antibody Fc domain adjacent thereto through a photoreactive amino acid. That is, the Fc regioselective binding peptide of the present invention can covalently bind to a specific functional group of the Fc domain through the photoreactive amino acid when irradiated with light.

In the present invention, the photoreactive amino acid is, for example, photoleucine, photomethionine, photoisoleucine, azidophenylalanine, azidohomoalanine, homopropargylglycine, homoallylglycine, p-acetyl-phenylalanine, p-pro It may be pargyloxy-phenylalanine, p-benzoyl-L-phenylalanine or cysteine to which benzophenone or aryl azide is bonded directly or through a linker, but is not limited thereto.

As one aspect of the present invention, the amino acid sequence of the Fc regioselective binding peptide may be SEQ ID NO: 13, but is not limited thereto.

For example, the Fc regioselective binding peptide in the present invention may be one in which the 10th amino acid position in the amino acid sequence of SEQ ID NO: 13 is substituted with an amino acid having a photoreactive functional group, but is not limited thereto.

Pseudomonas exotoxin A (PE) is a virulence factor of Pseudomonas aeruginosa, a Gram-negative bacterium that rarely infects healthy humans. PE belonging to the mono-ADP-ribosyl transferase family is a NAD ⁺ -diptamide-ADP-ribosyl transferase that exerts its cytotoxicity by inactivating elongation factor-2 (EF-2) for protein synthesis. PE is expressed as a protein with a length of 638 amino acids (aa) and can be divided into several structural and functional domains. In general, PEs belong to the family of two-component AB toxins, consisting of an A domain with enzymatic activity and a B domain with cell-binding subunits. Specifically, PE contains a very hydrophobic leader peptide of 25 aa at the N-terminus that is removed during secretion. The leader sequence is followed by the receptor binding domain Ia (aa 1-252), which consists of non-parallel β-sheets. Domain II (aa 253-364), with its six contiguous α-helices, allows toxins to move across cell membranes. The last four residues of domain Ib (aa 400-404) together with domain III (aa 405-613) form the catalytic subunit of the toxin with ADP-ribosyltransferase activity. PE catalyzes the inactivation of eukaryotic elongation factor 2 (eEF-2), an essential substance in the protein synthesis process, through ADP-ribosylation, and induces cell death through inhibition of protein synthesis.

In the present invention, "Pseudomonas exotoxin A (PE)" includes PE altered from a natural protein to reduce or eliminate immunogenicity. Such alterations may include, but are not limited to, removal of domain Ia, deletion of various amino acids in domains Ib, II and III, single amino acid substitutions, and addition of one or more sequences at the carboxyl terminus.

In one aspect of the invention, the altered PE may be a cytotoxic fragment of native wild-type PE. Cytotoxic fragments of PE may include fragments that exhibit cytotoxicity (eg, as proteins or pre-proteins) with or without subsequent proteolysis or other processing in the target cell. Specifically, the cytotoxic fragment of PE is at least about 20%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least 75%, more preferably at least about 90% of the cytotoxicity of native PE. % or more, even more preferably 95% cytotoxicity. More specifically, the cytotoxic fragment may have at least the cytotoxicity of native PE and, in some cases, increased cytotoxicity compared to native PE.

In the present invention, the modified PE may be, for example, PE4E, PE24, PE25, PE35, PE38, PE38QQR, PE38KDEL, PE40, or PE-LR, but is not limited thereto. As one aspect of the present invention, the PE may be PE24, and PE24 may be composed of, for example, amino acids represented by SEQ ID NO: 15, but is not limited thereto.

In the present invention, the Fc regioselective binding peptide containing photoreactive amino acids and Pseudomonas exotoxin A may be directly linked or indirectly linked through a peptide linker or the like.

In the present invention, the immunoconjugate may have an average drug-to-antibody ratio (DAR) of 1 to 10, specifically 1 to 5, 1 to 3, 1 to 2, or 2 It may be, but is not limited thereto.

In the present invention, "pharmaceutically acceptable salt" means a salt commonly used in the pharmaceutical industry, for example, inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc. and salts of inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, and acetylsalicylic acid. There are salts of organic acids such as and amino acid salts such as lysine, arginine, and guanidine. In addition, there are organic ion salts such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyl trimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification and separation processes. However, the types of salts meant in the present invention are not limited by these listed salts.

II. pharmaceutical composition

Another aspect of the present invention is a pharmaceutical composition for preventing or treating c-Kit-positive cancer, comprising the immunoconjugate or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect of the present invention is a method for treating or preventing c-Kit-positive cancer, comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof.

Another aspect of the present invention is the use of the immunoconjugate for preventing or treating c-Kit-positive cancer. Another aspect of the present invention is the use of the immunoconjugate for the preparation of the pharmaceutical composition.

The immunoconjugate of the present invention is as described above.

Since the immunoconjugate of the present invention induces cell death by specifically binding to c-Kit and inhibiting intracellular protein synthesis, it can be usefully used for treatment or prevention of c-Kit-positive cancer.

In the present invention, the c-Kit-positive cancer includes all cancers related to the c-Kit gene, and may be solid cancer or hematological cancer. For example, pseudomyxoma, intrahepatic bile duct cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myelogenous leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovarian Epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Barter cancer, bladder cancer, peritoneal cancer, Parathyroid cancer, adrenal cancer, mastocytoma, rhinosinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain cancer, childhood lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvic cancer, kidney cancer, heart Cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal , gastrointestinal stromal cancer, Wilms' cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, chorionic villus disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid carcinoma, vaginal cancer , spinal cord cancer, acoustic schwannoma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer , hematological cancer, and thymic cancer, and may be at least one selected from the group consisting of, specifically, mast cell tumor or gastrointestinal stromal tumor, but is not limited thereto.

In the present invention, the term "prevention" refers to any activity that suppresses or delays the progression of c-Kit-positive cancer disease by administration of the composition according to the present invention, and "treatment" refers to inhibition of the development of c-Kit-positive cancer disease. , means the alleviation or elimination of cancer disease.

The term "therapeutically effective amount" as used herein refers to an amount of the immunoconjugate effective for the treatment or prevention of cancer. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, type of disease, It may be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously in combination. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and the dosage can be easily determined by a person skilled in the art according to various factors such as the patient's condition, age, sex, and complications. can

Embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Furthermore, "include" a certain component throughout the specification means that other components may be further included without excluding other components unless otherwise stated.

The immunoconjugate of the present invention exhibits excellent anticancer activity by specifically binding to a specific region of c-Kit, inhibiting intracellular protein synthesis, and exhibiting cytotoxicity against c-Kit-positive cells.

1 is a graph showing the results of SPR analysis for confirming the c-KIT affinity of the 2G4 antibody.
Figure 2 shows the experimental results for confirming the c-KIT binding domain of the 2G4 antibody.
Figure 3 is the result of performing SDS-PAGE on the single conjugate or double conjugate of 2G4-PE immunoconjugate under non-reducing and reducing conditions.
Figure 4 shows the results of a comparative experiment confirming the c-Kit binding affinity of 2G4, 2G4-PE single immunoconjugate or 2G4-PE double immunoconjugate by ELISA. All results are expressed as the mean ± standard error of at least three independent experiments.
Figure 5 shows the results of a comparative experiment confirming the c-Kit binding affinity of 2G4, 2G4-PE single immunoconjugate or 2G4-PE double immunoconjugate by flow cytometry. All results are expressed as the mean ± standard error of at least three independent experiments.
6 shows the results of analyzing the cytotoxicity of 2G4-PE single immunoconjugate, 2G4-PE double immunoconjugate, or 2G4-DM1 in c-Kit-positive HMC-1.2 and GIST-48 cells in a dose-dependent manner. Results are expressed as the mean ± standard error of at least three independent experiments.
7 shows the results of analyzing the cytotoxicity of the 2G4-PE immunoconjugate in c-Kit-negative MDA-MB-453 cells in a dose-dependent manner. Results are expressed as the mean ± standard error of at least three independent experiments.
8 shows the results obtained by treating HMC-1.2, GIST-48, and MDA-MB-453 cells with 2G4, PE24, or 2G4-PE immunoconjugates and measuring inhibition of protein synthesis by Western blotting. Cycloheximide was used as a positive control and GAPDH was used as a loading control.
9 is a graph showing changes in tumor volume measured by intravenous administration of vehicle or 2G4 immunoconjugate or oral administration of imatinib in a mouse model transplanted with HMC-1.2 cells. Tumor volume was plotted according to the number of days after drug administration, green arrows indicate administration of vehicle or 2G4-PE immunoconjugate.
10 is a graph showing changes in tumor volume measured by intravenous administration of vehicle or 2G4 immunoconjugate or oral administration of imatinib in a mouse model transplanted with GIST-48 cells. Tumor volume was plotted according to the number of days after drug administration, green arrows indicate administration of vehicle or 2G4-PE immunoconjugate. Results are expressed as the mean ± standard error of at least three independent experiments and compared using an unpaired Student's two-sided t-test (compared to control; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared to Imatinib; ^# p < 0.05, ^## p < 0.001 compared to 2G4-PE 0.5 mg/kg; ^‡ p < 0.01).

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Example 1: Cell lines and culture conditions

The GIST cell line (GIST-48) was developed by Dr. It was provided by Sebastian Bauer (University of Duisburg-Essen, Germany). The human mast cell tumor cell line, HMC-1.2, was purchased from Millipore (MA, USA). A breast cancer cell line, MDA-MB-453, was purchased from American Type Culture Collection (MA, USA). GIST-48 cells were cultured in IMDM (Welgene, Daegu, Korea) containing 15% fetal bovine serum (FBS; Hyclone, UT, USA) and 1% penicillin/streptomycin (HyClone, UT, USA). 1.2 Cells were cultured in IMDM containing 10% FBS, 1% P/S and 50 μM 2-mercaptoethanol (Sigma, MO, USA), MDA-MB-453 cells were cultured in 10% FBS and 1% P/S. It was cultured in RPMI-1640 (Hyclone, UT, USA) containing S. All cell lines were maintained at 37 °C in a humidified 5% CO ₂ incubator.

Example 2: 2G4 Antibody Generation

The variable region of the 2G4 antibody was grafted onto the human Fc amino acid sequence and cloned into a pCHO vector (Life Technologies, CA, USA). The light chain variable region was fused in frame to the human kappa constant region, and the heavy chain variable region was fused in frame to the human IgG1 constant region. The leader peptide sequence for secretion of the full-length IgG1 antibody into the medium was added to the two genes to synthesize the gene, and then it was verified again through sequencing. Three clones were selected for expression testing in CHO cells. Glycerol stocks were prepared for the three clones, and endotoxin-free plasmids were prepared for expression testing in CHO cells.

The plasmid DNA was transfected into CHO-S cells. One week prior to transfection, CHO-S cells (Invitrogen, CA, USA) were transferred into monolayer cultures in DMEM-supplemented serum. And after dividing the cells 1 day before transfection, a nucleic acid-lipofectamine complex was prepared for the transfection sample and incubated overnight at 37°C in a 5% CO ₂ incubator. The culture was cultured for one week while adding the medium once every 2 to 3 days. Thereafter, the culture medium was recovered, bound to Protein A/G agarose (invitrogen, CA, USA), and washed with PBS. It was then eluted with 0.1 M glycine (pH 2.8) and neutralized with 1 M Tris-HCl (pH 8.0). After dialysis with PBS, it was stored at -70 °C.

The amino acid and nucleic acid sequences of the 2G4 antibody are as follows.

light chain CDR1 QSLLHSNGYN Y (SEQ ID NO: 1) CDR2 LGS (SEQ ID NO: 2) CDR3 MQALQTIT (SEQ ID NO: 3) light chain variable region DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTI TFGQGTRLEI K (SEQ ID NO: 4) whole light chain METDTLLLWV LLLWVPGSTG DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTI TFGQGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 5) atggaaacag acacactcct cctctgggtc ctcctcctct gggtcccagg cagcacagga gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactatc accttcggcc aagggacacg actggagatt aaacgtacgg tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagag cttca acaggggaga gtgttga (SEQ ID NO: 6) heavy chain CDR1 GFTFSRYG (SEQ ID NO: 7) CDR2 IWYDGTNK (SEQ ID NO: 8) CDR3 AREDWAEAFD M (SEQ ID NO: 9) heavy chain variable region QVQLVESGGG VVQPGRSLRL SCAASGFTFS RYGMHWVRQA PGKGLEWVAV IWYDGTNKDY TDSVRGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARED WAEAFDMWGQ GTTVTVSS (SEQ ID NO: 10) full heavy chain METDTLLLWV LLLWVPGSTG QVQLVESGGG VVQPGRSLRL SCAASGFTFS RYGMHWVRQA PGKGLEWVAV IWYDGTNKDY TDSVRGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARED WAEAFDMWGQ GTTVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN S GALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK (SEQ ID NO: 11) atggaaacag acacactcct cctctgggtc ctcctcctct gggtcccagg cagcacagga caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc tcctgtgcag cgtctggatt caccttcagt cgctatggca tgcactgggt ccgccaggct ccaggca agg ggctggagtg ggtggcagtt atatggtatg atggaactaa taaagactat acagactccg tgaggggccg attcaccatc tccagagaca attccaagaa cacgctgtat cttcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagaagat tgggctgagg cttttgatat gtgggg ccaa gggacaacgg tcaccgtctc ttcagcctcc accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc c agcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccct gagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg tcagcaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga cat cgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga (SEQ ID NO: 12)

Example 3: Expression and purification of the FcIII-PE24 construct

A plasmid (pSPEL571, pSPEL572, pSPEL524 or pSPEL573) encoding the FcIII-PE24 structure represented by SEQ ID NO: 18 having one amber codon in FcIII was introduced into E. coli BL21 (DE3) together with the other two plasmids, pSPEL155 and pSPEL168. transformed. The latter was used to inhibit misincorporation of p-benzoyl-L-phenylalanine (pBPA) into the proline position of the protein by overexpressing E. coli prolyl-tRNA synthetase (EcProRS). A single colony was picked and inoculated into 1 mL of 2 x YT medium. After overnight incubation at 37 °C, the culture was diluted 100-fold with 2 x YT and the cells were incubated at 37 °C until the optical density (OD) (600 nm) was 0.5. The expression of BpaRS and EcProRS was induced by adding 0.2% L-arabinose and 50 nM anhydrous tetracycline, respectively. When the OD (600 nm) reached 1.0, 1 mM isopropyl ß-D-1-thiogalactopyranoside and 1 mM pBPA (Alfa Aesar, USA) were added to the medium and the protein was expressed at 25 °C for 16 hours. made it After periplasmic fractionation, the FcIII V10BPA-PE24 protein (SEQ ID NO: 19) was purified using Ni ⁺⁺ -immobilizing resin (Clonetech Laboratories, USA) according to the manufacturer's recommendations. The protein solution was exchanged into 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl using a centrifuge (MWCO: 10 kDa, Merck, USA).

Example 4: Preparation of 2G4-PE immunoconjugates

10 μM of 2G4 antibody and 30 μg of FcIII V10BPA-PE24 were mixed with 1 x PBS (pH 7.4) in a 24-well plate and UV lamp for 2 hours at 4°C in the dark (Model: U01-133-194, Lklab, Korea) 365 nm ultraviolet light was irradiated to the mixture by using.

After UV irradiation, the 2G4-PE24 conjugate was purified using anion exchange chromatography on a Mono-Q column (GE Healthcare Life Science, USA) equilibrated with buffer A (20 mM phosphate, pH 7.9). The 2G4-PE24 conjugate was eluted with a gradient of buffers A and B (20 mM phosphate, 1 M NaCl, pH 7.9) (10 to 20% B for monoconjugate, 20 to 30% B for double conjugate).

Example 5: Enzyme-Linked Immunosorbent Assay (ELISA)

To compare the binding affinity of 2G4 and 2G4-PE, 1x phosphate-buffered saline (pH 7.2) containing 20 ng recombinant human CD117/c-Kit protein (Sino Biological, Beijing, China) was incubated at 4 °C overnight at 96- Well plates (Nunc, Roskilde, Denmark) were coated. Each well was blocked with 1 × PBS containing 5% BSA for 1.5 h at room temperature. Then, 4-fold serial dilutions of 2G4 or 2G4-PE were added to each well to a final concentration of 800 pM for 1 hour at room temperature. After incubation, wells were washed with 1 x PBS containing 0.1% Tween-20. Then, horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibody (1:2000, Thermo Fisher Scientific, MA, USA) was added for 1 hour at room temperature. Then, it was washed again and TMB-enzyme-linked immunosorbent assay (ELISA) solution (Thermo Fisher Scientific, MA, USA) was added for 5 minutes at room temperature. The reaction was terminated with 1 MH ₂ SO ₄ . Absorbance was measured at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany).

Example 6: Flow cytometry

Cells were placed on ice and blocked with 1x DPBS (Dulbecco's phosphate-buffered saline) containing 5% BSA for 1 hour. Cells (2×10 ⁵ ) were then placed on ice and treated with 2G4 or 2G4-PE (5 nM) for 1 hour. Cells were washed 3 times with 1 x DPBS containing 2% BSA. Cells were then stained with fluorescein isothiocyanate-conjugated goat anti-human IgG secondary antibody (0.3 μg/mL; Invitrogen, CA, USA) and left on ice for 1 hour. After washing three times, the cells were resuspended in 1 x DPBS containing 0.1% BSA. Fluorescent signals were analyzed using Cy-Flow Cube 6 (Sysmex Partec, Goerlitz, Germany).

Example 7: In vitro cytotoxicity assay

GIST-48 (8 x 10 ³ cells/well), HMC-1.2 (5 x 10 ³ cells/well) and MDA-MB-453 cells (5 x 10 ³ cells/well) were placed in a 96-well plate (Greiner Bio- One, Alphen aan den Rijn, Netherlands) and incubated with 2G4 or 2G4-PE at the indicated concentrations in a 5% CO ₂ incubator at 37 °C for 4 days. In addition, for a comparative experiment with 2G4-DM1, 2G4-DM1 immunoconjugate was prepared by conjugating DM1 and antibody through SMCC ( N -succinimidyl-4-( N -maleimidomethyl)cyclohexane-1-carboxylate), and GIST-48 ( 8 x 10 ³ cells/well) and HMC-1.2 (5 x 10 ³ cells/well) in the same manner as above. Cells were then stained with Hoechst 33342 (10 μM; Thermo Fisher Scientific, MA, USA) for 30 minutes at 37° C. and total cells were counted using a Celigo Imaging Cytometer (Nexcelom Bioscience, MA, USA).

Example 8: ADP-ribosylation activity assay

The ADP-ribosylation activity of PE was determined by measuring the transfer of ADP-ribose from biotinylated NAD ⁺ to EF-2 according to a published method (Proc Natl Acad Sci USA 109 (18): 6898-6903, 2012) did Wheat germ extract ( ^Promega , USA) and incubated for 1 hour at 37 °C. Biotinylated EF-2 was detected by Western blot using streptavidin HRP-conjugated secondary antibody (Thermo Fisher Scientific, MA, USA).

Example 9: Protein synthesis inhibition assay

To evaluate the inhibition of protein synthesis by 2G4-PE24, BONCAT (bioorthogonal noncanonical amino acid tagging) technology was used. HMC-1.2, GIST-48 (c-Kit positive) and MDA-MB-453 (c-Kit negative) cells were seeded in 6-well plates at a density of 5 x 10 ⁴ cells/well and incubated at 37 °C in a humidified 5 Incubated for 24 hours in a % CO ₂ incubator. Then, 0.1 nM 2G4, PE24 or 2G4-PE24 was added to the wells and the cells were incubated again at 37° C. for 20 hours. Then, 4 mM azidohomoalanine (AHA; Click Chemistry Tools, USA) was added to each well for 2 hours. Cells were washed with cold PBS containing 1 mM MgCl ₂ and 0.1 mM CaCl ₂ and trypsinized. In addition, cells were lysed using 1% SDS (w/v) and cell lysates were subjected to SDS-PAGE. For Western blotting, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and reacted with biotin-PEG4-alkyne (Click Chemistry Tools, USA) through a Cu(I) catalyzed click reaction. Biotinylated proteins were detected by Western blot using a streptavidin HRP-conjugated antibody.

Example 10: In vivo experiment

For the xenograft assay, HMC-1.2 cells (1 × ^10/100 μL/mouse) were seeded with 50% Matrigel (Corning, NY, USA) in 5-week-old female CB-17 SCID mice (Charles River Laboratories, Yokohama; Japan) was subcutaneously transplanted. GIST-48 (5 x 10 ⁶ / 100 μL/mouse) cells were transplanted into NMRI-nu mice (Janvier Laboratories, Saint-Berthevin Cedex, France). Tumor-generating mice were randomly assigned to treatment groups for efficacy studies. In vivo efficacy studies began when the tumor volume reached approximately 200 mm ³ .

Imatinib (Santa Cruz Biotechnology, CA, USA) was dissolved in distilled water and orally administered to mice once daily for 30 days (100 mg/kg). 2G4-PE was intravenously administered 3 times for 30 days at the indicated concentrations. Tumor size was measured twice weekly, and tumor volume was calculated using the formula:

Tumor volume = (4/3) Х π Х (length/2) Х (width/2) Х (depth/2).

All data were analyzed using GraphPad Prism 9.0 software (La Jolla, CA, USA). Results are expressed as mean ± standard error (SEM). Statistical significance was calculated using one-way ANOVA with unpaired Student's t-test or Tukey's test to compare differences between groups. Statistical significance was set at p < 0.05.

Experimental Example 1: Confirmation of binding affinity of 2G4 antibody

Surface Plasmon Resonance (SPR) was performed to confirm the binding ability of the 2G4 antibody to c-KIT. Human c-KIT (SEQ ID NO: 20; Elabscience, USA) 20 μg, mouse c-KIT (SB, Lot # LC05DE2304) 20 μg, rat c-KIT used for antibody production using SR7500DC (Reichert, USA) (SB, Lot#LC06SE1787) 20 μg was immobilized on a PEG (Reichert, USA) chip. Thereafter, 2G4 antibody was shed at different concentrations, and the KD value, which is the affinity for c-KIT, was analyzed using Scrubber2 program.

The KD value is a value obtained by dividing kd by ka, and the lower the value, the greater the binding ability to the corresponding target.

The results are shown in Figure 1. The 2G4 antibody exhibited strong affinity for human c-KIT with a KD value of about 2.8237 (±0.9) x 10 ⁻¹² M. Affinity for humans was the highest, followed by mice and rats.

Experimental Example 2: Domain Mapping

Deletion variants (Q26-P520, D113-P520 △domain I, A207-P520 △domain I - II, K310-P520 △domain I-III) of the human c-Kit gene (SEQ ID NO: 20; NM_000222) were added to the c-terminus. FLAG was tagged and transfected into HEK293. Then, it was secreted into the culture medium and purified using FLAG antibody beads (Sigma-Aldrich, USA). ELISA was then performed.

As shown in FIG. 2 , the 2G4 antibody did not recognize c-Kit when domain II was deleted, proving that the specific binding site for c-Kit of the 2G4 antibody was domain II.

Experimental Example 3: Generation and characterization of 2G4-PE immunoconjugate (2G4-PE)

2G4-PE was generated using an Fc-binding peptide (FcBP)-derived photocrosslinking reaction. Since FcBP can bind to the CH ₃ -CH ₂ interface region of IgG1 with high affinity (KD ~ 10 nM), valine, the 10th amino acid of FcBP, was substituted with pBPA, a non-natural photoreactive amino acid. A photoreactive FcBP-PE fusion protein was produced in E. coli using an orthogonal pair of tRNA and aminoacyl-tRNA synthetase. Then, single conjugates or double conjugates of 2G4-PE were generated through covalent bond formation using photoconjugation at 365 nm. As a result of SDS-PAGE analysis, it was confirmed that there was a size shift in the 2G4 antibody and FcBP-PE junction (FIG. 3).

Next, since conjugation of the linker-payload to the antibody can induce a conformational change that affects the target binding affinity, the present inventors compared the c-Kit binding affinity of the 2G4 antibody with that of 2G4-PE did ELISA (FIG. 4) and flow cytometry (FIG. 5) showed that the c-Kit binding affinity of the 2G4 antibody and the 2G4-PE conjugate was similar.

Experimental Example 4: In vitro cytotoxicity assay

In previous studies, 2G4-DM1 competitively binds c-Kit to SCF, inhibits SCF-mediated c-Kit signaling, and delays the G2/M phase cell cycle in various c-Kit-positive cancer cells, including small cell lung cancer and leukemia. 2G4-DM1 inhibited 2G4-DM1 in DM1-resistant cells, such as slow-growing c-Kit-positive gastrointestinal stromal tumors (GIST-48) or self-activating c-Kit mutant mast cell tumors (HMC-1.2). did not respond (Mol Oncol 16(6) : 1290-1308, 2022). Therefore, the present inventors compared the in vitro cytotoxicity of 2G4-PE and 2G4-DM1 using GIST-48 and HMC-1.2 cell lines.

Consistent with previous results, the IC ₅₀ of 2G4-DM1 in HMC-1.2 cells was 10.23 nM, but the IC ₅₀ value could not be determined in GIST-48 cells. In addition, the deletion mutant PE24 lacking the cell receptor binding domain failed to induce efficient cell death even at high concentrations. In contrast, 2G4-PE efficiently induced apoptosis with an IC ₅₀ of 50 pM or less in both HMC-1.2 and GIST-48 cells (FIG. 6). However, 2G4-PE did not induce apoptosis in MDA-MB-453, a c-Kit-negative breast cancer cell line, even at high concentrations (FIG. 7).

Next, since protein synthesis inhibition by PE24 is a major mechanism for inducing cancer cell apoptosis, protein synthesis after 2G4-PE treatment was evaluated using the BONCAT method. Cell lines were incubated with AHA, a methionine substitute, and AHA present in the newly synthesized protein was detected by Western blot. Treatment with 2G4 antibody or PE24 alone did not inhibit protein synthesis. However, 2G4-PE efficiently inhibited novel protein synthesis in c-Kit-positive HMC-1.2 and GIST-48 cells, but not in c-Kit-negative MDA-MB-453 cells (Fig. 8). This suggests that 2G4-PE specifically inhibits protein synthesis only in c-Kit-positive cancer cells.

Experimental Example 5: In vivo anti-tumor activity assay

The in vivo efficacy of 2G4-PE was evaluated using a mouse model xenografted with HMC-1.2 and GIST-48 cell lines. In the HMC-1.2 model, the 2G4-PE double conjugate showed 50% tumor growth inhibition at 3 mg/kg and reduced tumor weight by 50% (FIG. 9). In the GIST-48 model, both imatinib and the 2G4-PE single immunoconjugate significantly promoted tumor growth stagnation for about 56 days, but only 2G4-PE significantly reduced tumor weight in all experimental groups compared to the vehicle control group. (Fig. 10, left). In addition, the 2G4-PE double immunoconjugate promoted tumor retention for 56 days at 0.5 mg/kg, while inhibiting tumor growth below the starting tumor volume at 1.5 mg/kg and 3 mg/kg, and in all experimental groups, the vehicle control group The tumor weight decreased when compared to (Fig. 10, right). On the other hand, no weight loss was observed in all experimental groups.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (10)

An immunoconjugate represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]
Ab-(LD)p
In the above formula,
Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to sites D113 to P206 of SEQ ID NO: 20 in c-Kit;
L is an Fc regioselective binding peptide in which the 10th amino acid position in the Fc binding peptide of SEQ ID NO: 13 is substituted with a photoreactive amino acid;
The photoreactive amino acid is photoleucine, photomethionine, photoisoleucine, azidophenylalanine, azidohomoalanine, homopropargylglycine, homoallylglycine, p-acetyl-phenylalanine, p-propargyloxy-phenylalanine, or p- benzoyl-L-phenylalanine;
D is Pseudomonas exotoxin A; and
p is an integer from 1 to 10;
According to claim 1,
The anti-c-Kit antibody comprises a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 1, a light chain CDR2 of SEQ ID NO: 2, and a light chain CDR3 of SEQ ID NO: 3; and 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, an immunoconjugate or a pharmaceutically acceptable salt thereof.
According to claim 1,
The anti-c-Kit antibody comprises a light chain variable region of SEQ ID NO: 4 and a heavy chain variable region of SEQ ID NO: 10, an immunoconjugate or a pharmaceutically acceptable salt thereof.
According to claim 1,
The anti-c-Kit antibody comprises a constant region derived from human IgG1, an immunoconjugate or a pharmaceutically acceptable salt thereof.
delete delete According to claim 1,
The photoreactive amino acid is p-benzoyl-L-phenylalanine (pBPA), an immunoconjugate or a pharmaceutically acceptable salt thereof.
According to claim 1,
The Pseudomonas exotoxin A is any one or more selected from the group consisting of PE4E, PE24, PE25, PE35, PE38, PE38QQR, PE38KDEL, PE40 and PE-LR, an immunoconjugate or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for preventing or treating c-Kit-positive cancer, comprising the immunoconjugate of any one of claims 1 to 4, 7 and 8 or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 9,
The pharmaceutical composition for preventing or treating c-Kit-positive cancer, wherein the c-Kit-positive cancer is mast cell tumor or gastrointestinal stromal tumor.