KR102544135B1 - c-Kit targeting immunoconjugate - Google Patents

Abstract

The present invention relates to an immunoconjugate targeting c-Kit, specifically comprising an anti-c-Kit antibody or antigen-binding fragment thereof, a linker, and a drug moiety that specifically binds to a specific region of c-Kit. It relates to an immunoconjugate and a pharmaceutical composition for preventing or treating cancer comprising the immunoconjugate as an active ingredient. Since the antibody or antigen-binding fragment thereof of the present invention specifically binds to a specific region of c-Kit and can be internalized into cells, an immunoconjugate prepared by combining it with a drug-linker conjugate has excellent anticancer activity.

Description

c-Kit targeting immunoconjugate {c-Kit targeting immunoconjugate}

The present invention relates to an immunoconjugate targeting c-Kit, specifically comprising an anti-c-Kit antibody or antigen-binding fragment thereof, a linker, and a drug moiety that specifically binds to a specific region of c-Kit. It relates to an immunoconjugate and a pharmaceutical composition for preventing or treating cancer comprising the immunoconjugate as an active ingredient.

Lung cancer is a major cause of cancer-related death and is largely classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Among these, SCLC is particularly noteworthy as it accounts for 15 to 25% of all lung cancer types. SCLC, a neuroendocrine tumor, is distinguished from NSCLC by its rapid tumor growth, highly invasive nature, and early-developing extensive metastases. SCLC is distinctly different from small cell carcinoma except lung in terms of disease progression, prognosis and etiology. Without appropriate treatment, the life expectancy of patients with SCLC is less than 4 months, and the 5-year relative survival rate is still extremely low, although it has improved by 7% over the past few decades (S. Wang, et al., Survival changes in patients with small cell lung cancer). and disparities between different sexes, socioeconomic statuses and ages, Sci Rep 7 (2017) 1339).

A variety of molecular markers have been implicated in the pathogenesis and prognosis of SCLC. Paracline or autocrine signaling pathways have been widely used to explain deregulated SCLC growth. In addition, p53, Rb, NOTCH, MYC and PI3K are aberrantly mutated in SCLC. However, well-established aetiological factors such as EGFR-mutation in NSCLC have not been identified in SCLC to date.

SCLC has a highly aggressive pathway and is characterized by genomic instability, vascular proliferation, and high metastatic potential. Consequently, at the time of diagnosis, most SCLC patients already show metastatic spread outside the thorax, which often leads to early death. In addition, most SCLC patients who are current or former heavy smokers are more likely to have tumor mutations (C:G > A:T transversions, the most common type of base substitution). Additionally, overexpression of c-Kit was observed in 70% of SCLC patients.

The c-Kit proto-oncogene encodes a transmembrane tyrosine kinase growth factor receptor belonging to the platelet-derived growth factor receptor (PDGFR) family and is dysregulated in various cancers. Previous studies have reported that the expression of c-Kit with oncogenic mutations is aberrantly or upregulated in various cancers, resulting in SCF-independent c-Kit activation, which is the cause of aggressive cancers (H. Bougherara, et al., The aberrant localization of oncogenic kit tyrosine kinase receptor mutants is reversed on specific inhibitory treatment, Mol Cancer Res 7 (2009) 1525-1533).

Interestingly, various lines of evidence show that SCLC cell lines and tumors express the c-Kit receptor and SCF mRNA, suggesting that these gene products constitute an autocrine loop that mediates tumor cell survival and growth. Immunohistochemical staining showed that overexpression of c-Kit was found in 70% of SCLC patients.

Imatinib was developed to target BCR-ABL, PDGFR and c-Kit and is currently used to treat chronic myelogenous leukemia, acute lymphocytic leukemia, gastrointestinal stromal tumor (GIST) and hypereosinophilic syndrome. Various in vitro and in vivo studies have reported that imatinib has therapeutic efficacy for SCLC, but imatinib failed to demonstrate sufficient therapeutic efficacy in phase 2 clinical trials due to lack of desired response (G.K. Dy, et al. , A phase II trial of imatinib (ST1571) in patients with c-kit expressing relapsed small-cell lung cancer: a CALGB and NCCTG study, Ann Oncol 16 (2005) 1811-1816). Therefore, another approach targeting c-Kit in SCLC is needed.

Under this background, the present inventors prepared a novel human antibody targeting c-Kit, and confirmed that an immunoconjugate containing the antibody had excellent anticancer effects in c-Kit-positive cancers, thereby completing the present invention.

S. Wang, et al., Survival changes in patients with small cell lung cancer and disparities between different sexes, socioeconomic statuses and ages, Sci Rep 7 (2017) 1339 H. Bougherara, et al., The aberrant localization of oncogenic kit tyrosine kinase receptor mutants is reversed on specific inhibitory treatment, Mol Cancer Res 7 (2009) 1525-1533 G.K. Dy, et al., A phase II trial of imatinib (ST1571) in patients with c-kit expressing relapsed small-cell lung cancer: a CALGB and NCCTG study, Ann Oncol 16 (2005) 1811-1816

One object of the present invention is to provide an immunoconjugate comprising an anti-c-Kit antibody or antigen-binding fragment thereof, a linker and a drug moiety, or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating 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-(LD) _p

In the above formula,

Ab is an anti-c-Kit antibody or antigen-binding fragment thereof that specifically binds to regions from R49 to C186 of SEQ ID NO: 15 in c-kit;

L is a linker;

D is a drug moiety; 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 refers to a combination of an antibody or an antigen-binding fragment thereof that binds to a specific region of c-Kit, an optional linker, and an optional drug.

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.

c-Kit consists of five extracellular domains consisting of IgG like repeats, juxtamembrane and a cytoplasmic kinase domain including ATP-binding (TK1) and phosphotransferase (TK2) domains cleaved by kinase inserts. 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 anti-c-Kit antibody has a characteristic of specifically binding to a specific epitope of c-Kit, that is, a site from R49 to C186 of SEQ ID NO: 15. Here, the sequence described in SEQ ID NO: 15 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: 15 and the like by mutation or the like Even if they are partially different, they are all included in the scope of the present invention. In addition, even when some sequences from R49 to C186 of SEQ ID NO: 15 are added, substituted, or deleted, it should be understood that all of them 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 heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3; And a reference antibody comprising a light chain variable region including the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10 and human c-Kit to bind competitively to each other. can In another example, the antibody comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3; and a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10, 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, the antibody may include a heavy chain variable region represented by SEQ ID NO: 5 and a light chain variable region represented by SEQ ID NO: 12. In the present invention, it was confirmed for the first time that the antibody can be effectively used as a component of an ADC by confirming that the antibody has excellent internalization efficacy in cancer cell lines.

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, including, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (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.

As used herein, the term "linker" refers to any chemical moiety capable of linking an antibody or antigen-binding fragment thereof to a drug moiety, and may be a cleavable linker or a non-cleavable linker. can

"Cleavable linker" refers to a linker capable of releasing a drug into the cytoplasm by being cleaved in response to a specific environmental factor. The cleavable linker may be enzymatically cleaved or non-enzymatically cleaved, such as valine-citrulline-p-aminobenzyl carbamoyl (Val-Cit-PAB), alanine-phenylalanine-p-aminobenzyl carbamoyl (Ala- Phe-PAB), alanine-alanine-p-aminobenzyl carbamoyl (Ala-Ala-PAB), valine-alanine-p-aminobenzyl carbamoyl (Val-Ala-PAB), phenylalanine-lysine-p-amino Benzyl carbamoyl (Phe-Lys-PAB), alanine-alanine-glycine-p-aminobenzyl carbamoyl (Ala-Ala-Gly-PAB) or glycine-glycine-glycine-p-aminobenzyl carbamoyl (Gly -Gly-Gly-PAB) moiety, but is not limited thereto.

A “non-cleavable linker” is a linker that has relatively high plasma stability and is resistant to proteolysis. The non-cleavable linker is, for example, MCC (6-maleimidocaproic acid N-hydroxy succinimide ester), SMCC (N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), LC-SMCC (N-succinimidyl-4-(N-maleimidomethyl) -cyclohexane-1-carboxy-(6-amidocaproate)), KMUA (maleimidoundecanoic acid N-succinimidyl ester), GMBS (γ-maleimidobutyric acid N-succinimidyl ester), EMCS (c-maleimidocaproic acid N-hydroxysuccinimide ester), MBS ( m-maleimidobenzoyl-N-hydroxysuccinimide ester), AMAS (N-(α-maleimidoacetoxy)-succinimide ester), SMPH (succinimidyl-6-(β-maleimidopropionamido)hexanoate), SMPB (N-succinimidyl 4-(p-maleimidophenyl) butyrate), PMPI (N-(p-maleimidophenyl)isocyanate), STAB (N-succinimidyl-4-(iodoacetyl)aminobenzoate), SIA (N-succinimidyl iodoacetate), SBA (N-succinimidyl bromoacetate), SBAP (N-succinimidyl 3-(bromoacetamido)propionate), or a combination thereof, but is not limited thereto. For example, in the present invention, the non-cleavable linker may include N-hydroxysuccinimide ester, in which case the moiety reacts with the primary amine of lysine in the antibody to form an ADC can affect the target binding affinity of As a specific example, the non-cleavable linker in the present invention may be SMCC of Formula 2 below, but is not limited thereto.

[Formula 2]

In the present invention, the "drug moiety" is a component of an immunoconjugate comprising the c-Kit antibody of the present invention, and is used alone or in combination with the c-Kit antibody and the drug moiety to improve immunity. It refers to any compound that, when prepared and used as a conjugate, produces at least one or more synergistic effects. Such drug moieties include, but are not limited to, V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule inhibitors, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), inhibitor of nuclear export of protein CRM1, DPPIV inhibitor, proteasome inhibitor, inhibitor of mitochondrial phosphoryl transfer reaction, protein synthesis inhibitor, kinase inhibitor, CDK2 inhibitor, CDK9 inhibitor, kinesin inhibitor , HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, or combinations thereof. As a specific example, the drug moiety may be a maytansinoid compound such as DM1, DM3, and DM4, for example, mertansine (DM1) of Formula 3 below, but is not limited thereto.

[Formula 3]

Simple functional groups such as C _1-3 alkyl, hydroxy, halogen, or amine groups are added to the compounds of Formulas 2 and 3, or existing functional groups such as hydroxy, methyl, oxo, or halogen are converted into other functional groups. Even if it is substituted or removed, it is obvious that it is included in the scope of equivalents of the present invention as long as it shows the same efficacy as the immunoconjugate of the present invention.

In one embodiment, a linker-drug conjugate in which the SMCC linker and the DM1 drug moiety are bonded may have a structure represented by Chemical Formula 4 below.

[Formula 4]

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.

In the present invention, the immunoconjugate may have an average drug-to-antibody ratio (DAR) of 1 to 8, specifically 1 to 4, but is not limited thereto.

II. pharmaceutical composition

Another aspect of the present invention is a pharmaceutical composition for preventing or treating 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 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 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 exhibits cytotoxicity by specifically binding to c-Kit and releasing a drug, it can be usefully used for the treatment or prevention of cancer.

In the present invention, the cancer includes all cancers related to the c-Kit gene, and may be solid cancer or blood 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, 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, duodenum 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 interstitial cancer, Wilms' cancer , breast cancer, sarcoma, penile cancer, pharyngeal cancer, villus disease of pregnancy, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, 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 It may be one or more selected from the group consisting of, and is particularly useful for the treatment of small cell lung cancer (SCLC), but is not limited thereto. The cancer may be a c-Kit positive cancer.

The pharmaceutical composition may additionally be used in combination with other anticancer agent(s), and a synergistic effect may be expected thereby. An anticancer agent that can be used in combination with the immunoconjugate of the present invention may be, for example, lurbinectedin, carboplatin, etoposide, or a combination thereof, but is not limited thereto.

In the present invention, the term "prevention" refers to any activity that suppresses or delays the progression of cancer by administering the composition according to the present invention, and "treatment" refers to suppression of the development of cancer, reduction or elimination of cancer. it means.

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.

Since the antibody or antigen-binding fragment thereof of the present invention specifically binds to a specific region of c-Kit and can be internalized into cells, an immunoconjugate prepared by combining it with a drug-linker conjugate has excellent anticancer activity.

Figure 1 shows the results of FACS analysis by incubating SCLC cell lines with 4C9 antibody in a dose-dependent manner.
Figure 2 shows the results of FACS and Western blot analysis after incubation of NCI-H1048 cells transfected with control or c-Kit si-RNA with 4C9 antibody. α-Tubulin was used as a loading control.
Figure 3 is the result of confirming the binding site for c-Kit of the 4C9 antibody.
4 is a domain mapping result of the 4C9 antibody.
5 is a competitive ELISA result of the 4C9 antibody.
Figure 6 shows the results of Western blot analysis of phosphorylation of c-Kit, Akt and ERK after treating GIST-T1, NCI-H526 or NCI-H1048 cells with 4C9 at the indicated concentrations in the presence or absence of SCF. α-Tubulin was used as a loading control.
7 is a result of measuring the expression level of c-Kit by Western blot after treating each cell line with the 4C9 antibody at the indicated time point. α-Tubulin was used as a loading control.
8 shows the results of measuring the internalization of the 4C9 antibody into various SCLC cell lines using FACS.
9 is a result of comparing the optical absorbances of 4C9 and 4C9-DM1 at 252 nm and 280 nm.
10 shows the results of comparison between 4C9 and 4C9-DM1 in SDS-PAGE.
11 is a result of comparing the binding affinities of 4C9 and 4C9-DM1 to c-Kit using ELISA.
12 is a result of comparing the binding affinities of 4C9 and 4C9-DM1 to c-Kit using SPR.
13 is a result of a cytotoxicity test in c-Kit positive or negative SCLC cell lines. Results are expressed as the mean +/- standard error of at least three independent experiments.
FIG. 14 shows analysis using a Celigo Imaging Cytometer after incubation of SCLC cell lines with 4C9, IgG-DM1 or 4C9-DM1 for 24 and 48 hours ( ^** and ^*** vs. control; ^## and ^{## #} vs. IgG-DM1). Results are expressed as mean +/- error of at least three independent experiments, and means were compared using The unpaired Student's two-sided t -test ( ^** P < 0.01, ^*** P < 0.001, ^## P < 0.01, ^### P < 0.001).
Figure 15 shows changes in tumor volume and body weight upon intravenous administration of vehicle, 4C9, IgG-DM1, or 4C9-DM1 to mice or combined administration with chemotherapy in a tumor transplantation mouse model. Green arrows indicate administration of vehicle, IgG-DM1, 4C9 or 4C9-DM1, blue and red arrows indicate administration of rubinectidine and carboplatin, respectively (*, ** and *** vs. their respective Corresponding vehicle control; ^§ and ^§§§ vs. their respective corresponding 4C9-DM1 control; ^† vs. carboplatin/etoposide; ^‡ vs. rubinectidine). Results are expressed as mean +/- standard error, and means were compared using The unpaired Student's two-sided t -test ( ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^## P < 0.01, ^### P < 0.001, ^† P < 0.05, ^‡ P < 0.01, ^§ P < 0.05, ^§§§ P < 0.001).

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-T1) was developed by Dr. It was provided by Sebastian Bauer (University of Duisburg-Essen, Germany). SCLC cell lines (NCI-H446, NCI-H526, NCI-H889, NCI-H1048 and NCI-H2170) and breast cancer cell lines (MDA-MB-453) were purchased from ATCC (VA, USA). All cell lines were cultured according to the manufacturer's recommendations and maintained at 37 °C in a humidified 5% CO ₂ incubator.

Example 2: 4C9 Antibody Generation

The nucleotide sequence of the 4C9 clone was codon optimized for the Chinese hamster (Cricetulus griseus), synthesized as IgG1, and subcloned into the pCHO 1.0 vector (Thermo Fisher Scientific, MA, USA). The recombinant plasmid was transiently transfected into CHO cells cultured in CD CHO serum-free medium using the ExpiCHO™ Expression System Kit (Thermo Fisher Scientific, MA, USA) for expression of the 4C9 antibody. Antibodies were purified using Protein A Sepharose columns and SP Sepharose columns (Invitrogen, CA, USA) according to previous studies (J.O. Kim, et al., Development and characterization of a fully human antibody targeting SCF/c-kit signaling, Int J Biol Macromol 159 (2020) 66-78).

The nucleotide and amino acid sequences of the 4C9 antibody are as follows.

CDR1 CDR2 CDR3 VH SYYWS (SEQ ID NO: 1) YIFYSGSTNY NPSLKS (SEQ ID NO: 2) GYSSGWLDFH H (SEQ ID NO: 3) [Signal Peptide: MKHLWFFLLL VAAPRWVLS (SEQ ID NO: 4)]
QVQLQESGPG LVKPSETLSL TCTVSGGSIG SYYWSWIRQP PGKGLEWIGY IFYSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCARGYS SGWLDFHHWG QGTLVAVSS (SEQ ID NO: 5) [atgaaacatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcc (SEQ ID NO: 6)]
aga (SEQ ID NO: 7) VL RASQSISSYL N (SEQ ID NO: 8) AASSLQS (SEQ ID NO: 9) QQSYSTPIT (SEQ ID NO: 10) [Signal Peptide: MRVPAQLLGL LLLWLRGARC (SEQ ID NO: 11)]
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPITFGQ GTRLEIK (SEQ ID NO: 12) [atgagggtcc ccgctcagct cctggggctc ctgctactct ggctccgagg tgccagatgt (SEQ ID NO: 13)]
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca aggttcagtg gcagtggat (SEQ ID NO: 14)

Example 3: Flow cytometry

Flow cytometric analysis was performed to confirm the binding of 4C9 to c-Kit on the cell surface. Cells (2.0 Х 10 ⁵ ) were placed on ice and blocked with 1 Х DPBS containing 5% BSA for 1 hour. Cells were then incubated for 1 h on ice with the indicated concentrations of 4C9 antibody. After washing three times with 1 Х DPBS containing 2% BSA, the cells were placed on ice and stained with 0.3 μg/ml FITC-conjugated anti-human IgG secondary antibody (Invitrogen, CA, USA) for 1 hour. Fluorescence was analyzed using a CyFlow Cube 6 (Sysmex Partec, Goerlitz, Germany).

Example 4: Western blot analysis

For SCF/c-Kit signal analysis, cells were left serum-free for 30 minutes. Cells were then pre-treated with 4C9 antibody at the indicated concentrations for 30 min. Next, 100 ng/ml recombinant human SCF (R&D Systems, Minneapolis, Minn., USA) was added and incubated for an additional 5 minutes. Cells were washed twice with DPBS and resuspended in RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM _NaEDTA , 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate). , 0.1% SDS, 10 mM β-glycerophosphate, 1 mM Na ₃ OV ₄ , 10 mM NaF, 1 μg/ml leupeptin, 1 mM PMSF, 5 μg/ml aprotinin ( aprotinin) and 2 mM 2-mercaptoethanol). Protein extracts were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). The membrane was blocked for 1 hour with 1 Х TBST containing 5% BSA at room temperature and blotted using the following antibodies as probes: anti-c-Kit (R&D Systems, MN, USA), anti-pc-Kit (Tyr568 /570, Tyr703, Tyr719 and Tyr823; Cell Signaling Technology, MA, USA), anti-Erk1/2 (Santa Cruz Biotechnology, CA, USA), anti-p-Erk1/2 (Cell Signaling Technology, MA, USA), anti-Akt (Santa Cruz Biotechnology, CA, USA), anti-p-Akt (Ser473; Cell Signaling Technology, MA, USA) and anti-α-tubulin (produced in our laboratory).

Example 5: siRNA test

For siRNA testing, cells were seeded in 150 mm ² plates. After 24 hours, the culture medium was replaced with Opti-MEM medium (Gibco, CA, USA). Cells were then incubated for 72 h with control siRNA or c-Kit siRNA (200 pmol; Santa Cruz Biotechnology, CA, USA) using Lipofectamine RNAiMAX reagent (Invitrogen, MA, USA) according to the manufacturer's instructions. transfected. c-Kit knockdown was confirmed by flow cytometry and Western blot analysis.

Example 6: Enzyme-Linked Immunosorbent Assay (ELISA)

A competitive ELISA was performed to determine whether the 4C9 antibody interfered with the binding of SCF to c-Kit. Recombinant human SCF protein (100 ng/well; R&D Systems, MN, USA) was coated on 96-well plates overnight at 4°C. Each well was blocked with 1 Х PBS containing 5% BSA for 2 h at room temperature. 4C9 antibody was pre-incubated with recombinant human c-Kit protein (2 μg/ml; Sino Biological, Beijing, China) at the indicated concentrations for 1 hour at room temperature. The mixture was added to each well for 1 hour at room temperature. Plates were washed 4 times with 1 Х PBS containing 0.1% Tween-20 and incubated for 1 hour with goat anti-c-Kit antibody (200 ng/ml; R&D Systems, Minn., USA). After washing, HRP-conjugated anti-goat IgG secondary antibody (1:2000; Santa Cruz Biotechnology, TX, USA) was treated for 1 hour. Then, a 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine) solution (Thermo Fisher Scientific, MA, USA) was added for 15 minutes. Signals were analyzed at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany). In addition, the binding affinities of 4C9 and 4C9-DM1 were compared using ELISA.

Example 7: Internalization analysis of 4C9 antibody

Cells were incubated with cycloheximide (75 μg/ml) to prevent translocation of de novo synthesized c-Kit to the cytoplasmic membrane. Then, cells were incubated with human BD Fc block ^TM (BD Biosciences, San Diego, CA, USA) and 5% BSA (CellNest, Gyeonggi-do, Korea) for 10 min at room temperature to inhibit Fc receptor-mediated internalization of antibodies. Blocked. Cells were washed with PBS and incubated with or without 4C9 antibody (1 μg/ml) at 4° C. or 37° C. for 1-4 hours. Cells were then incubated with FITC-conjugated anti-human IgG (0.3 μg/mL, Invitrogen) for 1 hour, washed 3 times with PBS and analyzed using CyFlow ^® Cube 6 (PARTEC).

Example 8: Generation of ADC

First, the 4C9 antibody and normal human IgG1 (Sino Biological, Beijing, China) were dialyzed against a conjugation buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). Antibody and linker-drug conjugate, SMCC-DM1 (MedChemExpress, NJ, USA) were mixed in a 1:5 molar ratio and the mixture was spun at room temperature for 90 minutes. To remove aggregated ADC, the product was centrifuged at 20,000 g and filtered through a Supor (Hydrophilic polyethersulfone) membrane (0.2 μm, Pall Life Sciences, NY, USA). ADC was purified by size exclusion chromatography (Superdex ^TM 200, GE Healthcare, WI, USA) and stored in storage buffer (10 mM sodium succinate, 0.05% polysorbate 20 and 6% sucrose; pH 5.0). Dialyzed. The DAR (drug-antibody ratio) of 4C9-DM1 was determined by comparing the absorbance of the naked antibody and ADC at 252 nm because DM1 absorbs ultraviolet light at 252 nm, and liquid chromatography mass spectrometry was used. analyzed through In addition, the molar concentration of linked DM1 was determined by UV spectroscopy utilizing different absorbance maxima of 4C9 and SMCC-DM1 at 280 nm and 252 nm, respectively:

A 280 = εAb280 Х CAb + εD280 Х CD (1a)

A 252 = εD252 Х CD + εAb252 Х CAb (1b)

here:

εD280 = Molecular extinction coefficient of SMCC-DM1 at 280 nm.

εAb280 = Molecular extinction coefficient of 4C9 at 280 nm.

εD252 = Molecular extinction coefficient of SMCC-DM1 at 252 nm.

εAb252 = Molecular extinction coefficient of 4C9 at 252 nm.

CD = molar concentration of SMCC-DM1.

CAb = molar concentration of 4C9.

Divide equation (1b) by (1a) and rearrange to get equation (2).

DAR = CD/CAb = (εAb252 - RεAb280) / (RεD280 - εD252) (2)

where R is the total absorbance ratio (A252/A280). An optical path length of 10 mm was assumed using these equations. SMCC-DM1 molar extinction coefficients (εD280 = 5,065 M ^-1 cm ^-1 and εD252 = 21,168 M ^-1 cm ^-1 ) were determined experimentally in our laboratory. In the case of 4C9, the value estimated using the ExPASy ProtParam tool for the extinction coefficient is εAb280 = 223,400 M ^-1 cm ^-1 , and the value experimentally measured in our laboratory is εAb252 = 77,117 M ^-1 cm ^-1 .

Spectra were recorded using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, DE, USA). Samples were diluted to about 4.5 mg/ml in formulation buffer. A blank was recorded using the same buffer and measured at ambient temperature.

Example 9: Cytotoxicity assay

Cells (3 to 7 Х 10 ³ cells) were seeded in a 96-well plate and cultured for 3 to 5 days with 4C9 or 4C9-DM1 at the indicated concentrations according to the growth rate of each cell line. Live cells were then stained with 10 µM Hoechst 33342 (Thermo Fisher Scientific, MA, USA) for 30 minutes and counted using a Celigo Imaging Cytometer (Nexcelom, MA, USA).

Example 10: Cell cycle analysis

For cell cycle analysis, 7-10 Х 10 ³ cells were seeded in 96-well plates and incubated with 1 μg/ml 4C9, 4C9-DM1 or IgG1-DM1 for 24, 48 and 72 hours. Cells were fixed with 2% paraformaldehyde for 10 min at 4 °C (for suspension cell lines) or with 80% cold ethanol at 4 °C for at least 2 h (for adherent cell lines). After washing twice with DPBS, cells were stained with 50 μg/ml propidium iodide solution (Sigma, MO, USA; containing 0.1 μg/ml RNase A and 0.05% Triton X-100) at 37°C for 90 min. did Fluorescence was analyzed using a Celigo Imaging Cytometer.

Example 11: Investigation using a xenograft mouse model

Based on the in vitro cytotoxicity assay, the in vivo efficacy of 4C9-DM1 was investigated using a mouse model xenografted with NCI-H526. All animal studies were approved by the Ajou University Animal Care Committee (IACUC 2019- 0005), and all experiments were conducted in accordance with appropriate regulations and guidelines. 5-week ^- old female CB-17 mice (Orientbio ^, Seongnam; Korea) was subcutaneously injected into the right flank. Drug administration was started when the tumor volume reached about 200 mm ³ . Naked antibody and ADC were administered intravenously at the indicated concentrations on days 0, 7 and 14. Rubinectidine (0.09 mg/kg; MedChemExpress, NJ, USA) was administered intravenously on the day following ADC infusion (Days 1, 8 and 15). Carboplatin (60 mg/kg; Sigma, MO, USA) was administered intraperitoneally on days 1 and 11, and etoposide (3 mg/kg; Sigma, MO, USA) was administered on days 1 to 5 and 11 to 15. It was administered intraperitoneally on the 1st. Tumor volume was measured twice a week using a Vernier caliper, and the volume calculation formula used was as follows:

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

Experimental Example 1: Confirmation of c-Kit specific binding of 4C9 antibody

First, the present inventors confirmed whether the 4C9 antibody prepared in Example 2 could specifically bind to c-Kit. FACS analysis showed that 4C9 binds to various SCLC cell lines including NCI-H526, NCI-H1048 and NCI-H889 in a dose-dependent manner. Interestingly, binding of the 4C9 antibody was saturated at 50 ng/ml to c-Kit positive SCLC cell lines. As previously reported, c-Kit expression was higher in NCI-H889 cell lines than in NCI-H526 and NCI-H1048 cells. However, 4C9 did not show any cross-reactivity with c-Kit-negative SCLC cell lines NCI-H446 and NCI-H2170 cells (FIG. 1).

The present inventors additionally analyzed c-Kit specific binding of 4C9 through siRNA knockdown experiments. Western blot analysis using 4C9 confirmed that c-Kit siRNA efficiently reduced protein expression in NCI-H1048 cells. In addition, FACS analysis using 4C9 confirmed that c-Kit expression on the cell surface was down-regulated by c-Kit siRNA (FIG. 2). Taken together, it was shown that 4C9 binds specifically to the extracellular domain of c-Kit on the cell surface.

Experimental Example 2: Domain Mapping

Deletion mutants (Q26-P520, D113-P520 Ddomain I, A207-P520 Ddomain I-II, K310-P520 Ddomain I-III, T411-P520 Ddomain I-IV) of the human c-Kit gene (NM_000222, SEQ ID NO: 15) was tagged with FLAG at the c-terminus and transfected into HEK293. Then, the protein was secreted into the culture medium, purified using FLAG antibody beads (Sigma-Aldrich), and ELISA was performed. The results are shown in Figure 3. According to FIG. 3 , the 4C9 antibody did not recognize c-Kit when domain I was deleted, indicating that the binding site for c-Kit of the antibody was domain I.

Next, deletion mutants (Q26-P520, R49-P520, I70-P520, K100-P520, K116-P520, N130-P520, S187-P520) of the human c-kit gene (NM_000222, SEQ ID NO: 15) were c- HEK293 was transfected by tagging FLAG at the end. Then, the protein was secreted into the culture medium, purified using FLAG antibody beads (Sigma-Aldrich), and ELISA was performed.

The results are shown in FIG. 4 . According to FIG. 4, the antibody was found to bind from R49 of domain I to C186 of domain II.

On the other hand, competitive ELISA results showed that the binding of c-Kit to SCF was reduced in a 4C9 antibody concentration-dependent manner (FIG. 5). These results indicate that the 4C9 antibody competitively binds to c-Kit with SCF.

Experimental Example 3: Analysis of suppression of SCF-mediated phosphorylation of c-Kit and evaluation of c-Kit stability

We analyzed whether 4C9 could inhibit SCF-mediated phosphorylation of c-Kit. In the GIST-T1 cell line, pre-treatment with 4C9 antibody decreased c-Kit phosphorylation in a dose-dependent manner. However, the phosphorylation levels of ERK and Akt, which are downstream molecules of c-Kit, were not changed by 4C9 antibody treatment. Interestingly, total c-Kit levels were reduced by 4C9 antibody treatment (Fig. 6). Therefore, a decrease in the level of phosphorylated c-Kit may be due to a decrease in the expression level of c-Kit.

On the other hand, as a result of c-Kit stability evaluation, the 4C9 antibody dramatically reduced the total c-Kit level in a time-dependent manner in the GIST cell line, but slightly decreased it in the SCLC cell line (FIG. 7), which correlated with ubiquitination-dependent degradation. there may be Unlike GIST-T1, 4C9 did not reduce SCF-mediated c-Kit phosphorylation or c-Kit stability in NCI-H526 and NCI-H1048 cell lines. In addition, the 4C9 antibody did not inhibit the phosphorylation of ERK and Akt induced by SCF (FIG. 6), suggesting that the 4C9 antibody cannot be applied as an antagonist of SCF/c-Kit signaling.

Experimental Example 4: Creation and Characterization of ADC (4C9-DM1)

Despite the overexpression of c-Kit in SCLC cell lines, the contribution of SCF/c-Kit signaling in the pathogenesis of SCLC is limited and in light of the failure of imatinib, anti-c-Kit naked antibodies are applicable for SCLC treatment (Clin Cancer Res., 2003, 9: 5880; Ann Oncol., 2005, 16: 1811). Therefore, the present inventors thought that using ADC for SCLC treatment could be a reasonable alternative. Since one of the most important factors to be considered when applying ADC is the efficacy of antibody internalization after forming a complex with a target molecule, the present inventors first tried to confirm whether the 4C9 antibody is internalized in SCLC cell lines.

As a result of FACS analysis, the internalization efficacy of 4C9 antibody was 91% in NCI-H526, 76.6% in NCI-H1048 and 68.6% in NCI-H889 cells (Fig. This indicates that it can be used as an efficient carrier for specific delivery.

Next, the present inventors prepared an ADC containing the 4C9 antibody using a non-cleavable linker, SMCC, and a microtubule inhibitor, DM1. Since DM1 absorbs ultraviolet light at 252 nm, the absorbances of the naked antibody (4C9) and ADC (4C9-DM1) were measured and compared at 252 nm. As a result, the absorbance of 4C9-DM1 at 252 nm was higher than that of 4C9 (FIG. 9). The DAR was found to be about 2.16.

In addition, 4C9 and 4C9-DM1 were compared using SDS-PAGE. 10 μg of each protein was loaded on SDS-PAGE, stained with Coomassie Brilliant Blue G250 and destained with 50% methanol. As a result, SDS-PAGE analysis showed that SMCC-DM1 conjugation to 4C9 antibody induced slight size fluctuations (FIG. 10).

Next, since the N-hydroxysuccinimide ester of the SMCC linker reacts with the primary amine of lysine in the antibody, it can affect the target binding affinity of the ADC. ELISA analysis demonstrated similar binding affinities of 4C9 and 4C9-DM1 to c-Kit (FIG. 11). Quantitative analysis of binding using surface plasmon resonance (SPR) showed that the binding affinity of 4C9 to human c-Kit was 5.5 Х 10 ^-9 M (Ka = 2.27 Х 10 ⁴ M ^-1 s ^-1 and Kd = 1.27 Х 10 ^-4 s ^-1 ), and 4C9-DM1 was also confirmed to show similar binding affinity (5.46 Х 10 ^-9 M; Ka = 2.14 Х 10 ⁴ M ^-1 s ^-1 and Kd = 1.16 Х 10 ^-4 s ^-1 ) (FIG. 12).

Taken together, these results indicate that conjugation to the 4C9 antibody with SMCC-DM1 did not affect the binding affinity of the 4C9 antibody to c-Kit.

Experimental Example 5: In vitro antitumor activity assay

Next, the inventors used various SCLC cell lines (NCI-H526, NCI-H889, NCI-H1048, NCI-H446 and NCI-H2170) and a breast cancer cell line (MDA-MB-453) to evaluate 4C9-DM1 in vitro. Cytotoxicity was assayed. c-Kit negative cell lines (NCI-H446, NCI-H2170 and MDA-MB-453) served as negative controls.

As a result, 4C9-DM1 exhibited cytotoxicity against c-Kit-positive NCI-H526, NCI-H889 and NCI-H1048 with an IC ₅₀ value ranging from 158 pM to 4 nM. These results represent a 4- to 300-fold increase compared to the activity against c-Kit-negative cancer cell lines (FIG. 13 and Table 2).

IC ₅₀ value (nM) c-Kit expression organization type cell line 4C9-DM1 ^* SMCC-DM1 c-Kit positive SCLC NCI-H526 0.158 12.23 NCI-H889 0.323 11.62 NCI-H1048 4.08 30.45 c-Kit Voice SCLC NCI-H446 16.58 6.97 NCI-H2170 35.50 20.17 breast cancer MDA-MB-453 47.63 9.763

* The molar concentration was calculated as the molecular weight of 4C9-DM1 at 180 kDa.

Interestingly, the cytotoxic activity of DM1 against c-Kit-positive cancer cells was 7- to 77-fold higher when applied as ADC than when applied as payload alone. In contrast, the cytotoxic activity against c-Kit negative cancer cells was 2- to 5-fold lower (Table 2). These results indicate that off-target toxicity can be reduced when the payload is applied as an ADC.

Next, DM1 inhibits microtubule assembly, induces cell cycle arrest in the G2/M phase, and induces apoptosis in actively dividing cells. Therefore, the effect of 4C9-DM1 on the cell cycle was analyzed using the c-Kit-positive SCLC cell line NCI-H526 and the c-Kit-negative SCLC cell line NCI-H446.

As a result, 4C9-DM1 significantly increased the G2/M phase cell population over time, but 4C9 and IgG-DM1 did not. In addition, 4C9, 4C9-DM1 and IgG-DM1 did not affect the G2/M phase population in c-Kit negative NCI-H446 cells (FIG. 12). The above results indicate that cell cycle arrest in NCI-H526 cells is specifically mediated through DM1 delivered by complex formation between 4C9-DM1 and c-Kit.

Experimental Example 6: In vivo antitumor activity assay

Based on the above in vitro cytotoxicity assay, the in vivo effect of 4C9-DM1 was tested using a mouse model xenografted with NCI-H526. As a result, there was no effect on IgG-DM1, but 4C9-DM1 significantly inhibited the growth of NCI-H526 tumors in a dose-dependent manner (FIG. 15). At doses of 1 mg/kg, 3 mg/kg, and 5 mg/kg, the tumor growth inhibition (TGI) rates of 4C9-DM1 were 35%, 45%, and 60%, respectively, compared to the vehicle control group. The 4C9 antibody alone also partially inhibited tumor growth, but this result was not statistically significant. On the other hand, no weight loss by the administered substance was observed, confirming that there is no concern about toxicity.

Next, chemotherapy including etoposide, cisplatin, carboplatin, and ruvinectidine is used as standard therapy for treating patients with SCLC. Combination therapy using chemotherapy drugs is effective, but most SCLC relapses rapidly within 1 year. Therefore, the present inventors tried to confirm whether combination chemotherapy using 4C9-DM1 can improve the therapeutic effect on SCLC.

As a result, 4C9-DM1, rubinectidine and carboplatin/etoposide exhibited similar antitumor activity. The TGI ratio of these groups was at the 50% level compared to the vehicle control group on day 18. Interestingly, the combination of 4C9-DM1 with ruvinectidine or carboplatin/etoposide synergistically inhibited tumor growth. The TGI rates of both groups were found to be at the 85% level compared to the vehicle control group with subsequent regrowth on day 21. The combination treatment of 4C9-DM1 and rubinectidine resulted in a weight loss of about 10%, but it was confirmed that the weight increased again after treatment was discontinued (FIG. 15). This indicates that the combination can be effectively used to treat SCLC with manageable or mild toxicity.

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 (11)

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 regions from R49 to C186 of SEQ ID NO: 15 in c-Kit;
The anti-c-Kit antibody
a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 2, and the heavy chain CDR3 of SEQ ID NO: 3; and
a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10;
L is MCC (6-maleimidocaproic acid N-hydroxy succinimide ester), SMCC (N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), LC-SMCC (N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy- (6-amidocaproate)), KMUA (maleimidoundecanoic acid N-succinimidyl ester), GMBS (γ-maleimidobutyric acid N-succinimidyl ester), EMCS (c-maleimidocaproic acid N-hydroxysuccinimide ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide) ester), AMAS (N-(α-maleimidoacetoxy)-succinimide ester), SMPH (succinimidyl-6-(β-maleimidopropionamido)hexanoate), SMPB (N-succinimidyl 4-(p-maleimidophenyl)butyrate), PMPI (N- (p-maleimidophenyl)isocyanate), STAB (N-succinimidyl-4-(iodoacetyl)aminobenzoate), SIA (N-succinimidyl iodoacetate), SBA (N-succinimidyl bromoacetate) and SBAP (N-succinimidyl 3-(bromoacetamido)propionate) Any one or more selected from the group consisting of, non-cleavable linker (non-cleavable linker);
D is a maytansinoid drug moiety; and
p is an integer from 1 to 8;
delete delete The method of claim 1, wherein the anti-c-Kit antibody
Comprising a heavy chain variable region represented by SEQ ID NO: 5, and a light chain variable region represented by SEQ ID NO: 12,
An immunoconjugate or a pharmaceutically acceptable salt thereof.
delete delete delete delete A pharmaceutical composition for preventing or treating c-Kit-positive cancer, comprising the immunoconjugate of claim 1 or 4 or a pharmaceutically acceptable salt thereof as an active ingredient.
delete The pharmaceutical composition for preventing or treating cancer according to claim 9, further comprising at least one selected from the group consisting of lurbinectedin, carboplatin, and etoposide.

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