WO2024090916A1 - Pharmaceutical composition for cancer treatment comprising anti-igsf1 antibody - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating colon cancer, biliary tract cancer, or head and neck cancer, comprising an anti-IGSF1 antibody as an active ingredient. When the anti-IGSF1 antibody according to the present invention was treated while co-culturing colon cancer cells, biliary tract cancer cells, or head and neck cancer cells with immune cells, the secretion of Granzyme B, and cytokines IFN-γ and TNF-α was increased. It was confirmed that the growth of colon cancer or biliary tract cancer was inhibited when the antibody was administered to a xenogeneic or allogeneic colon cancer or biliary tract cancer implantation mouse model. Therefore, the anti-IGSF1 antibody of the present invention can be helpful in the treatment of colon cancer, biliary tract cancer, and head and neck cancer.

Description

Pharmaceutical composition for cancer treatment comprising anti-IRGSF1 antibody

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an anti-IGSF1 antibody or fragment thereof as an active ingredient.

Cancer is one of the greatest diseases threatening human health. It is a disease that occurs when cells undergo a series of mutations, proliferate in an unlimited and uncontrolled manner, and become immortal. Causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital genetic mutations. For cancer discovered in the early stages, there are treatments such as surgery, radiation therapy, and chemotherapy, but their side effects are becoming a major problem, and for terminal cancer or metastatic cancer, life is terminal and ends without any special treatment.

Among these cancers, in the case of colon cancer, epidermal growth factor receptor (EGFR) such as FOLFIRI (Folinic acid, Fluorouracil and Irinotecan), FOLFOX (Folinic acid, Fluorouracil and Oxaliplatin), Erbitux, Vectibix, etc. Receptor-targeted anticancer antibody treatments and angiogenesis inhibitors such as Avastin or Zaltrap are mainly used as anticancer treatments. However, the treatment response rate in patients who have received a prescription is very low at about 20%, or the recurrence rate within 3 years is high in patients who have shown treatment effects, and the drug is showing limited effectiveness, such as lack of response to the drug or resistance occurring after treatment. .

Biliary tract cancer has the characteristic that surgical resection is not effective due to its high recurrence rate and that it does not respond well to conventional chemotherapy or radiotherapy. Biliary tract cancer is treated as a first-line treatment with Gemcitabine, Capecitabine, or 5-Fluorouracil (5-FU) as single agents, or with Oxaliplatin or Cisplatin. It is recommended to use it in combination with platinum compounds such as ), or in combination with gemcitabine and capecitabine. However, multi-agent chemotherapy does not show lasting effects in patients with metastatic and/or recurrent biliary tract cancer, and in the case of advanced biliary tract cancer, the 5-year survival rate after diagnosis is less than 5%.

Additionally, for head and neck cancer, surgery, radiation therapy, and chemotherapy are mainly used. Among these, cisplatin, a representative anticancer drug for the treatment of head and neck cancer, has excellent anticancer effects, but in the case of patients with cisplatin resistance or patients who develop resistance during the treatment process, problems to overcome resistance are needed. am.

Accordingly, the present inventors studied a method for effectively treating cancer and completed the present invention by confirming that tumor growth was inhibited by administration of an anti-IGSF1 antibody or fragment thereof.

In order to solve the above problem, one aspect of the present invention is to prevent or treat any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer, comprising an anti-IGSF1 antibody or fragment thereof as an active ingredient. A pharmaceutical composition is provided.

Another aspect of the present invention provides a method for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer, comprising administering an anti-IGSF1 antibody or fragment thereof to a subject.

Another aspect of the present invention provides an anti-IGSF1 antibody or fragment thereof for use in the prevention or treatment of any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer.

Another aspect of the present invention provides the use of an anti-IGSF1 antibody or fragment thereof for the manufacture of a medicament for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer.

When the anti-IGSF1 antibody according to the present invention was treated with colon cancer cells, biliary tract cancer cells, or head and neck cancer cells and immune cells, the secretion of Granzyme B and the cytokines IFN-γ and TNF-α increased. . It was confirmed that the growth of colon cancer or biliary tract cancer was inhibited when the antibody was administered to a xenograft or allogeneic colon cancer or biliary tract cancer implanted mouse model. Therefore, the anti-IGSF1 antibody of the present invention can be usefully used in the treatment of colon cancer, biliary tract cancer, and head and neck cancer.

Figure 1 is a diagram showing the results of confirming the expression of IGSF1 in human colon cancer cell line HT29 cells through flow cytometry.

Figure 2 is a graph showing the results of confirming changes in the secretion amount of Granzyme B, IFN-γ, and TNF-α by treatment with WM-A1-3389 antibody in a co-culture system of HT29 cells and human peripheral blood cells (PBMC).

Figure 3 is a graph showing the results of measuring the degree of tumor growth by administration of WM-A1-3389 antibody in a mouse model implanted with the human colon cancer cell line HT29.

Figure 4 is a diagram showing the results of confirming the expression of IGSF1 in the mouse colon cancer cell line MC38 through immunofluorescence staining (top) and flow cytometry (bottom).

Figure 5 is a graph showing the results of measuring the degree of tumor growth by administration of WM-A1-3389 antibody in a mouse model implanted with the mouse colon cancer cell line MC38.

Figure 6 is a diagram showing the results of confirming the expression of IGSF1 in the mouse colon cancer cell line CT26 through immunofluorescence staining (top) and flow cytometry (bottom).

Figure 7 is a graph showing the results of measuring the degree of tumor growth by administration of WM-A1-3389 antibody in a mouse model implanted with the mouse colon cancer cell line CT26.

Figure 8 is a diagram showing the results of confirming the expression of IGSF1 in human biliary tract cancer cell line Choi-CK cells through flow cytometry.

Figure 9 is a graph showing the results of confirming changes in the secretion amount of Granzyme B, IFN-γ, and TNF-α by treatment with WM-A1-3389 antibody in a co-culture system of Choi-CK cells and human peripheral blood cells (hPBMC).

Figure 10 is a graph showing the results of measuring the degree of tumor growth by administration of WM-A1-3389 antibody in a mouse model implanted with the human biliary tract cancer cell line Choi-CK.

Figure 11 is a diagram showing the results of confirming the expression of IGSF1 in the human head and neck cancer cell line FaDu cells through flow cytometry.

Figure 12 is a graph showing the results confirming changes in the secretion amounts of Granzyme B, IFN-γ, and TNF-α by treatment with WM-A1-3389 antibody in the FaDu cell and human peripheral blood cell (hPBMC) co-culture system.

anti-IGSF1 antibody

As used herein, the term “IGSF1” is a membrane protein encoded by the IGSF1 gene found on the X chromosome of humans and other mammalian species. Although the function of IGSF1 in normal cells is not well known, IGSF1 mutations are known to cause diseases such as IGSF1 deficiency syndrome or central hypothyroidism.

In the present invention, the IGSF1 may be included without limitation as long as it is mammalian IGSF1, but preferably refers to human IGSF1. Additionally, in the present invention, the IGSF1 protein includes, but is not limited to, both native and mutant IGSF1 proteins. The native IGSF1 protein generally refers to a polypeptide containing the amino acid sequence of the native IGSF1 protein, and the amino acid sequence of the native IGSF1 protein generally refers to the amino acid sequence found in naturally occurring IGSF1. Information about IGSF1 can be obtained from known databases such as GenBank of the National Institutes of Health, and may have, for example, an amino acid sequence (SEQ ID NO: 19) with Genbank accession number NP_001164433.1, but is not limited thereto.

As used herein, the term “antibody” refers to an immunoglobulin molecule that reacts immunologically with a specific antigen, and refers to a protein molecule that specifically recognizes the antigen. The antibodies include whole antibodies, monoclonal antibodies, polyclonal antibodies, single domain antibodies, single chain antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs, Fab fragments, F (ab') fragment, disulfide bond-linked Fv (sdFv), and epitope-binding fragments of any of the above.

As used herein, the term “anti-IGSF1 antibody” refers to an antibody capable of specifically binding to IGSF1, and may be used interchangeably with “IGSF1-specific antibody” in the present invention. In particular, anti-IGSF1 antibodies can specifically bind to the C terminus of IGSF1. The form of the antibody may include both whole antibodies and antibody fragments thereof.

The heavy and light chains of immunoglobulins may each include a constant region and a variable region. The light and heavy chain variable regions of immunoglobulins include three variable regions called complementarity determining regions (CDRs) and four framework regions (FRs). The CDR mainly functions to bind to the epitope of the antigen. The CDRs of each chain are typically called CDR1, CDR2, and CDR3 sequentially, starting from the N terminus, and are identified by the chain on which the specific CDR is located.

The anti-IGSF1 antibody or fragment thereof of the present invention includes H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3. It may include a heavy chain variable region (VH). In addition, the anti-IGSF1 antibody or fragment thereof of the present invention includes L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L- CDR2 containing the amino acid sequence of SEQ ID NO: 6. It may include a light chain variable region (VL) including CDR3. At this time, the heavy chain variable region may include the amino acid sequence of SEQ ID NO: 7, and the light chain variable region may include the amino acid sequence of SEQ ID NO: 8. In this specification, the antibody may be referred to as WM-A1-3389.

The heavy chain variable region of the antibody consists of the amino acid sequence of SEQ ID NO: 7 and about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about It may comprise or consist of an amino acid sequence having at least 97%, at least about 98%, at least about 99%, or 100% identity. In addition, the light chain variable region of the antibody is about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, and about 96% or more of the amino acid sequence of SEQ ID NO: 8. , may include or consist of an amino acid sequence having an identity of at least about 97%, at least about 98%, at least about 99%, or 100%.

Immunoglobulin heavy chain constant regions (CH) exhibit different amino acid compositions and sequences and therefore possess different types of antigenicity. Accordingly, immunoglobulins can be divided into five categories and referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. Additionally, depending on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds, the same type of Ig can be classified into different subtypes. For example, IgG can be classified as IgG1, IgG2, IgG3, and IgG4. Light chains can be classified as κ or λ chains depending on their different constant regions. Each of the five types of IgG can have either a κ or λ chain.

When the anti-IGSF1 antibody or fragment thereof of the present invention includes a constant region, it may include a constant region derived from IgG, IgA, IgD, IgE, IgM, or a partial hybrid thereof.

As used herein, the term “hybrid” means that sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins exist within the single-chain immunoglobulin heavy chain constant region. For example, a domain hybrid consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, and CH3 of IgG, IgA, IgD, IgE, and IgM is possible.

Additionally, when the anti-IGSF1 antibody or fragment thereof of the present invention includes a light chain constant region (LC), the light chain constant region may be derived from a λ or κ light chain.

As used herein, the term “antibody fragment” refers to an scFv fragment, which is an Fv fragment that binds to IGSF1, as well as a Fab fragment, Fab' fragment, and F(ab') ₂ fragment having antigen-binding activity, and is used in the present invention. Contains the CDR regions of the antibodies described in. The Fv fragment is the smallest antibody fragment that contains the heavy and light chain variable regions, without constant regions, and retains all antigen-binding sites.

pharmaceutical composition

The pharmaceutical composition containing the anti-IGSF1 antibody or fragment thereof of the present invention as an active ingredient exhibits preventive or therapeutic efficacy against any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer.

At this time, the colon cancer, biliary tract cancer, and head and neck cancer may be cancers in which IGSF1 is overexpressed.

The term “colorectal cancer” used in the present invention refers to a malignant tumor composed of cancer cells that arise in the large intestine (cecum, appendix, colon, rectum, or anal canal). Pathologically, colon cancer is mostly adenocarcinoma, but it also includes lymphoma, sarcoma, squamous cell carcinoma, and metastatic lesions of other cancers. By site, it is largely divided into colon cancer and rectal cancer, with the highest incidence of cancer occurring in the lower colon, or rectum, at about 50%. The cause of colon cancer is still unclear, but genetic factors, dietary habits related to the consumption of high-fat and low-fiber foods, and inflammatory bowel disease are being considered. Colon cancer can occur at any age, but the incidence increases with age and occurs frequently in people in their 50s or 60s. The incidence ratio between men and women is somewhat higher for colon cancer in women and for rectal cancer in men.

The term “biliary tract cancer” used in this specification is also called cholangiocarcinoma and refers to cancer that occurs in the bile ducts. The bile duct is a tube that transports bile produced in the liver to the duodenum. Cancers originating in the bile duct are divided into intrahepatic bile duct cancer (about 20% to about 25%), hilar bile duct cancer (about 50% to about 60%), and distal liver cancer, depending on the anatomical location. It can be classified into bile duct cancer (about 20% to about 25%). Adenocarcinoma arising from cholangiocytes accounts for the majority of cholangiocarcinomas. The cause of cholangiocarcinoma is not yet clearly known, and it is caused by chronic inflammation in the bile duct cells lining the bile ducts, bile duct stones, sclerosing cholangitis, liver dystomatosis, inflammatory bowel disease, and bile duct cysts caused by congenital dilatation of the bile ducts. It is known to be related to etc.

As used herein, the term "head and neck cancer" refers to malignant tumors that occur in the face, nose, throat, mouth, larynx, pharynx, salivary glands, and thyroid gland, excluding tumors that occur in the brain and eyes. Among head and neck cancers, cancer that occurs in the oral cavity is called oral cancer, cancer that occurs in the larynx, the organ that produces sound, is called laryngeal cancer, and cancer that occurs in the pharynx is called pharyngeal cancer. The pharyngeal cancer is divided into nasopharyngeal cancer, oropharyngeal cancer, and hypopharyngeal cancer depending on the location of occurrence. In addition, head and neck cancer includes cervical esophageal cancer, salivary gland cancer, salivary gland cancer, malignant lymphoma, malignant melanoma, and various soft tissue cancers, and thyroid cancer is included in head and neck cancer in a comprehensive sense. Smoking and drinking are known to be traditional causes of head and neck cancer, and nasopharyngeal cancer is known to be closely related to viral infection (Epstein Barr virus). In addition, gastric acid reflux disease, esophageal disease, radiation or ultraviolet rays, vitamin or iron deficiency, etc. are known to be factors that increase the incidence of head and neck cancer.

As used herein, the term “prevention” refers to all actions that suppress or delay the onset of a disease by administering the pharmaceutical composition. The term “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by administering the pharmaceutical composition.

In the pharmaceutical composition of the present invention, the anti-IGSF1 antibody or fragment thereof may be included in any amount (effective amount) depending on use, formulation, formulation purpose, etc., as long as it can exhibit anti-cancer activity. Here, “effective amount” refers to the amount of active ingredient that can induce an anticancer effect. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art. The pharmaceutical composition of the present invention contains the antibody as an active ingredient in an amount of about 0.1% to about 90% by weight, specifically about 0.5% by weight to about 75% by weight, and more specifically about 1% by weight to about 1% by weight, based on the total weight of the composition. It can be contained at 50% by weight.

Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Accordingly, “enhanced efficacy” (e.g., improvement in efficacy) may be due to improved pharmacokinetic parameters and improved efficacy, compared to clearance rates and tumor growth in test animals or human subjects, or to survival, recurrence rates, or disease progression. It can be measured by comparing parameters such as survival in the absence of it.

As used herein, the term "efficacy" means survival over a period of time, such as 1 year, 5 years, or 10 years, or disease-free survival, which can be determined by one or more parameters. You can. Additionally, the parameter may include that the size of at least one tumor in the subject is suppressed.

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

The pharmaceutically acceptable carrier may be any carrier that is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. Pharmacologically acceptable adjuvants (buffers, dispersants) may also be included in the pharmacological composition.

As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as sweeteners, solubilizers, wetting agents, emulsifiers, isotonic agents, absorbents, antioxidants, preservatives, lubricants, fillers, buffers, and bacteriostatic agents are added as needed. can do.

The compositions of the present invention can be prepared in a variety of formulations for parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection). When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. Injectable preparations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerogeratin, etc. can be used. Meanwhile, injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

Regarding the formulation of pharmaceutical compositions, it is known in the art, and specifically, references can be made to the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The above documents are considered part of this specification.

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

As used herein, the term "administration" means introducing a predetermined substance into an individual by an appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, locally, intranasally, or rectally, but is not limited thereto.

Here, “therapeutically effective amount” or “pharmaceutically effective amount” refers to the amount of a compound or composition effective in preventing or treating the target disease, which is sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment. This refers to an amount that does not cause side effects. The level of the effective amount is determined by factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and It may be determined based on other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of drug effective in treating the disease.

Specifically, the dosage of the composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally administered from about 0.1 mg to about 1,000 mg per kg of body weight, or from about 5 mg to about 200 mg per kg of body weight every day or every other day. Alternatively, it can be administered at intervals of 2 to 3 weeks, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the scope of the present invention is not limited thereto.

The term "subject" refers to an object to which the composition of the present invention can be applied (prescribed), including humans, rats, mice, guinea pigs, hamsters, rabbits, dogs, cats, chickens (including eggs), pigs, monkeys, It may be a mammal such as a goat. Preferably, it may be a human, but is not limited thereto.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. At this time, the other therapeutic agent may additionally include any compound or natural extract whose safety has already been verified and which is known to have anticancer activity in order to increase or reinforce the anticancer activity.

Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect with minimal or no side effects, and this can be easily determined by a person skilled in the art.

Another aspect of the present invention is to treat any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer, comprising administering to an individual a pharmaceutical composition containing an anti-IGSF1 antibody or fragment thereof as an active ingredient. Provides prevention or treatment methods for Here, anti-IGSF1 antibody and fragment thereof, pharmaceutical composition, colorectal cancer, biliary tract cancer, head and neck cancer, administration, treatment and prevention are the same as described above.

The subject may be a mammal, preferably a human. Additionally, the individual may be a patient with the disease or an individual who is likely to suffer from the disease.

The administration route, dosage, and frequency of administration of the pharmaceutical composition may be administered to the subject in various methods and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency of administration can be determined by a person skilled in the art. You can select by range. Additionally, the anti-IGSF1 antibody or fragment thereof may be administered in combination with other drugs or biochemically active substances known to have therapeutic effects on the disease, or may be formulated in the form of a combination preparation with other drugs.

Another aspect of the present invention is the prevention or treatment of any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer using a pharmaceutical composition containing the anti-IGSF1 antibody or fragment thereof of the present invention as an active ingredient. Provides a purpose. The anti-IGSF1 antibody and fragment thereof, pharmaceutical composition, colon cancer, biliary tract cancer, head and neck cancer, treatment and prevention are the same as described above.

Another aspect of the present invention provides an anti-IGSF1 antibody or fragment thereof for use in the prevention or treatment of any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer. The anti-IGSF1 antibody and fragment thereof, pharmaceutical composition, colon cancer, biliary tract cancer, head and neck cancer, treatment and prevention are the same as described above.

Another aspect of the present invention provides the use of an anti-IGSF1 antibody or fragment thereof for the manufacture of a medicament for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer. Here, the anti-IGSF1 antibody and fragment thereof, pharmaceutical composition, colon cancer, biliary tract cancer, head and neck cancer, prevention and treatment are the same as described above.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1. Production of anti-IGSF1 antibody

Example 1.1. IGSF1 antigen expression and purification

After amplifying only the extracellular domain of IGSF1 from the Jurkat cell cDNA library through PCR, human Fc (fragment crystallizable region) and Histack were added to the carboxy terminus (C terminus) using the N293F vector (Y Biologics Co., Ltd.) An IGSF1 protein expression vector was created by fusing a polynucleotide encoding hig tag). HEK293F cells were transfected with the prepared IGSF1 expression vector and then cultured for 6 days in medium supplemented with 1 mM valporic acid (valproate). Then, after primary purification of the IGSF1 extracellular domain using protein A agarose, secondary purification of the IGSF1 extracellular domain using Superdex 200 gel filtration chromatography, It was used for antibody selection.

Example 1.2. Selection of IGSF1 human antibodies

After coating and blocking the IGSF1 antigen, bio-panning (bio-panning, Y Biologics Co., Ltd.) was performed using the prepared human antibody library phage (Y Biologics Co., Ltd.), and the antigen-specific binding was performed. Only phages were eluted. The second and third rounds of biopanning were performed using the phage amplified in the first round of biopanning. ELISA was performed to confirm the antigen specificity of the positive phage antibody pool obtained through each round of biopanning. In addition, it was confirmed that anti-IGSF1 antibody was enriched in the phage pool obtained through the third round. In each polyphage ELISA, hundreds of monoclones were selected from the third round of panning with high binding capacity, and using these, preliminary antibody clones were obtained by confirming whether they specifically bind to IGSF1 through ELISA analysis. . For the selected preliminary antibody clones, 99 phages with different base sequences were selected through DNA sequence analysis. It was confirmed that the 99 selected positive phage clones strongly bound to the antigen, IGSF1, but did not bind to other antigens. Through the above method, a total of 95 types were selected as a result of selecting antibodies showing specificity for the IGSF1 antigen using various other antigens.

Example 1.3. Confirmation of specificity for IGSF1 antigen

The specificity of the selected antibodies to other antigens, including IGSF1, was compared and analyzed using ELISA. The binding of phage clones to various types of unspecified antigens, including control antigens mFc, hRAGE-Fc, CD58-Fc, and ITGA6-Fc, was confirmed. The antibodies obtained in this way were converted from phage to IgG whole vectors, and it was confirmed that the heavy and light chain sequences of the converted 95 clones matched the sequences of the phage antibodies. Among the obtained antibodies, the most optimized antibody was selected and named “WM-A1-3389”. The CDR sequence of the WM-A1-3389 antibody is shown in Table 1 below.

CDR	amino acid sequence	sequence number
H-CDR1	GGTFSTYA	One
H-CDR2	IIPFVGTV	2
H-CDR3	VRDGRSYFDS	3
L-CDR1	TSNIGSNL	4
L-CDR2	DNH	5
L-CDR3	VAWDDSLNGYV	6

Example 1.4. WM-A1-3389 antibody production

To produce the WM-A1-3389 antibody, a polynucleotide (SEQ ID NO: 23) encoding the heavy chain (SEQ ID NO: 21) was loaded into the N293F vector (Y Biologics Co., Ltd.) (hereinafter referred to as HC DNA). In addition, a polynucleotide (SEQ ID NO: 24) encoding the light chain (SEQ ID NO: 22) was loaded into the N293F vector (Y Biologics Co., Ltd.) (hereinafter referred to as LC DNA). After the vector was transformed into cells, WM-A1-3389 antibody was obtained and purified. Purified proteins were confirmed through SDS-PAGE.

	amino acid sequence	sequence number
Heavy chain	QVQLVQSGAEVKRPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGRIIPFVGTVDYAQKFQDRVTITADKSTNTAYMELSSLRSEDTAVYYCVRDGGRSYFDSWGPGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	21
Light chain	QFVLTQPPSVSAAPGQDVIISCSGNTSNIGSNLVSWFQQFPETAPKLLIYDNHKRPSGISDRFSGTKSGTSASLAISGLQSEDEADYYCVAWDDSLNGYVFGTGTKVTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC	22

Example 2. Anticancer activity of WM-A1-3389 antibody against colon cancer

Example 2.1. Confirmation of IGSF1 expression in human colon cancer cell lines

First, the expression of IGSF1 was confirmed in HT29 cells, a human colon cancer cell line.

Specifically, the medium of HT29 cells was removed, washed once with PBS, and then treated with 2 ml of Accutase to separate the cells. The separated cells were diluted in 8 ml of PBS (hereinafter referred to as FACS buffer) containing 2% (v/v) FBS and 0.05% (w/v) sodium azide, then centrifuged at 1,200 rpm for 1 minute to remove the supernatant. did. The cell pellet was released with a vortex and resuspended with an appropriate amount of FACS buffer, and the number of cells was counted using trypan blue and distributed to each tube to reach 1Х10 ⁵ cells. Then, the supernatant was removed by centrifugation at 1,200 rpm for 1 minute, and the cell pellet was released using a vortex. Then, 0.4 ㎍ of Alexa Fluor 647-labeled anti-human IGSF1 antibody (sc-393786 AF647, Santa Cruz Biotechnology) was added per 200 ㎕ of FACS buffer and incubated at 4°C for 30 minutes, protected from light. After the reaction was completed, 1 ml of FACS buffer was added to each tube and centrifuged at 1,200 rpm for 1 minute to remove the supernatant. The above process was performed a total of two times. Finally, 200 ㎕ of FACS buffer was added to the remaining cell pellet, resuspended, and analyzed by FACS. For FACS analysis, the fluorescence value of Alexa 647 labeled in each cell was measured using BD LSRFortessa Flow cytometry (BD Bioscience), and the results were analyzed using FLowJo software.

As a result, as shown in Figure 1, the proportion of cells expressing IGSF1 in the HT29 cell line was approximately 49.8%.

Example 2.2. Confirmation of changes in cytokine secretion by co-culture of human colon cancer cell lines and peripheral blood cells

Using a co-culture system of colon cancer cell lines and peripheral blood cells, changes in cytokine secretion due to treatment with WM-A1-3389 antibody were confirmed.

Specifically, human-derived peripheral blood cells (hPBMC) were centrifuged at 1,200 rpm for 10 minutes to remove the supernatant, and resuspended in 10% RPMI. Afterwards, HT29 colon cancer cells and hPBMC were inoculated at a 1:1 ratio, and 1 ㎍/㎖ SEB (Sigma), 10 ㎍/㎖ IgG (Bio The cells were treated and cultured in a carbon dioxide incubator at 37°C for 24 hours. At this time, IgG was used as a negative control. The supernatant of the co-cultured cells was collected in a 1.5 ml Eppendorf tube and the amount of cytokine secretion was measured through Cytometric bead array (CBA) assay. The CBA assay used a BD Biosciences product and was performed according to the BD Biosciences protocol. Standard (standard sample) and co-cultured cell supernatant were added to each tube, capture beads were added, and reaction was performed at room temperature for 1 to 2 hours. After reaction, PE detection reagent was added and further reaction was performed at room temperature for 2 hours. Each tube was washed with washing buffer and measured using LSRFortessa™ Flow Cytometry equipment (BD Bioscience).

As a result, as shown in Figure 2, the secretion amount of cytokines IFN-γ and TNF-α, including Granzyme B, in HT29 colon cancer cells expressing IGSF1 was statistically significantly lower in the group treated with the WM-A1-3389 antibody compared to the control group. It was confirmed that there was a significant increase.

Example 2.3. Confirmation of anticancer activity of WM-A1-3389 antibody in xenogeneic colon cancer cell line transplantation mouse model

The anticancer activity of the WM-A1-3389 antibody was confirmed in a colon cancer mouse model transplanted with a human colon cancer cell line.

Specifically, 6-week-old female peripheral blood mononuclear cell humanized mice (Gem biosciences) were purchased and acclimatized for 1 week, and then HT29 cells, a human colon cancer cell line, were diluted in PBS and instilled on the right ventral side to a concentration of 5Х10 ⁶ cells/mice. It was injected subcutaneously (100 ㎕). Human IgG isotype was used as a negative control for the WM-A1-3389 antibody. When the tumor size reached an average of 140 mm ³ to 160 mm ³ , human IgG isotype (negative control) was administered intraperitoneally at a dose of 10 mg/kg or WM-A1-3389 antibody at a dose of 30 mg/kg. Administration was conducted once every three days for four weeks, and the tumor size and body weight of the mice were measured twice a week. On the 28th day of administration, the experimental animals were euthanized and the tumors were extracted and weighed.

As a result, as shown in Figure 3, the WM-A1-3389 antibody administration group showed higher tumor growth inhibition efficacy than the negative control group.

When compared to the negative control group, the WM-A1-3389 antibody dosage and tumor growth inhibition (TGI) are listed in Table 3 below (***: P<0.001).

	WM-A1-3389 antibody administration group
Dose (mg/kg)	30
TGI (%)	55.5 ± 4.4
P-Value	0.0002 (***)

As shown in Table 3, tumor growth inhibition (TGI) was shown to be about 55.5%. In addition, it was confirmed that tumor growth was suppressed in individual subjects.

Example 2.4. Confirmation of anticancer activity of WM-A1-3389 antibody in allogeneic colon cancer cell line transplantation mouse model

Example 2.4.1. Confirmation of IGSF1 expression in mouse colon cancer cell line MC38

The expression of IGSF1 in MC38 cells, a mouse colon cancer cell line, was confirmed through immunofluorescence staining and flow cytometry. Immunofluorescence staining was performed as follows. Cells with the supernatant removed were fixed with 4% (v/v) formaldehyde (PFA, T&I) at room temperature for 15 minutes. The fixed cells were washed twice with PBS, blocking buffer was added to prevent non-specific binding, and reacted at room temperature for 1 hour. After the reaction, the primary IGSF1 antibody (sc-393786, Santa Cruz Biotechnology) was treated at a ratio of 1:100 and reacted at 4°C for more than 12 hours, protected from light. After the reaction, the cells were washed twice with PBS, treated with anti-mouse IgG H&L (Alexa 488) (ab150113, Abcam) antibody as a secondary antibody at a ratio of 1:100, and incubated for 1 hour at room temperature, shielded from light. The stained cells were washed twice with PBS and observed under a fluorescence microscope (Olympus). Flow cytometry was performed in the same manner as Example 2.1.

As a result, as shown in Figure 4, expression of IGSF1 was observed in MC38 cells, and the proportion of cells expressing IGSF1 was approximately 45.4%.

Example 2.4.2. Confirmation of anticancer activity of WM-A1-3389 antibody in mouse colon cancer cell line MC38 implanted mouse model

The anticancer activity of the WM-A1-3389 antibody was confirmed in a colon cancer mouse model (MC38 syngeneic mouse model) transplanted with MC38 cells, a mouse colon cancer cell line.

Specifically, 5-week-old female C57BL/6N mice were purchased and acclimatized for 1 week. Mouse colon cancer cells MC38 cells were diluted in PBS to prepare a concentration of 2.5Х10 ⁵ cells/mice, and MC38 cells were injected subcutaneously (100 ㎕) into the right ventral side of the mouse. Mouse IgG isotype was used as a negative control for the WM-A1-3389 antibody. When the tumor size reached an average of 50 mm ³ to 100 mm ³ , mouse IgG isotype (negative control) was administered intraperitoneally at doses of 10 mg/kg and WM-A1-3389 antibody at doses of 3, 10, and 30 mg/kg, respectively. Administration was performed once every three days for two weeks, and the tumor size and body weight of the mice were measured twice a week. On the 14th day of administration, the experimental animals were euthanized and the tumors were extracted and weighed.

As a result, as shown in Figure 5, the WM-A1-3389 antibody administered group showed higher tumor growth inhibition efficacy than the negative control group, and a correlation was confirmed in which the tumor growth inhibition rate (TGI) increased in a dose-dependent manner. . When compared to the negative control, the WM-A1-3389 antibody dosage and tumor growth inhibition rate (TGI) are listed in Table 4 below.

	WM-A1-3389 antibody administration group
Dose(mg/kg)	3	10	30
TGI(%)	50.8 ± 4.3	58.2 ± 4.8	68.7 ± 3.8

Example 2.4.3. Confirmation of IGSF1 expression in mouse colon cancer cell line CT26

The expression of IGSF1 in CT26 cells, a mouse colon cancer cell line, was confirmed through immunofluorescence staining and flow cytometry. Immunofluorescence staining was performed in the same manner as in Example 2.4.1, and flow cytometry was performed in the same manner as in Example 2.1.

As a result, as shown in Figure 6, expression of IGSF1 was observed in the CT26 cell line, and the proportion of cells expressing IGSF1 was approximately 57.2%.

Example 2.4.4. Confirmation of anticancer activity of WM-A1-3389 antibody in mouse colon cancer cell line CT26 implanted mouse model

The anticancer activity of the WM-A1-3389 antibody was confirmed in a colon cancer mouse model (CT26 syngeneic mouse model) transplanted with CT26 cells, a mouse colon cancer cell line.

Specifically, 5-week-old female C57BL/6N mice were purchased, acclimatized for 1 week, and mouse colon cancer CT26 cells were diluted in PBS and injected subcutaneously (100 ㎕) into the right ventral side to a concentration of 2.5Х10 ⁵ cells/mice. did. Human IgG isotype was used as a negative control for the WM-A1-3389 antibody. When the tumor size reached an average of 80 mm ³ to 100 mm ³ , 10 mg/kg of mouse IgG isotype (negative control) and WM-A1-3389 antibody were administered intraperitoneally at doses of 3, 10, and 50 mg/kg, respectively. . Administration was performed once every three days for two weeks, and the tumor size and body weight of the mice were measured twice a week. On the 14th day of administration, the experimental animals were euthanized, the tumors were extracted, and their weight was measured.

As a result, as shown in Figure 7, the WM-A1-3389 antibody administered group showed higher tumor growth inhibition efficacy than the negative control group, and a correlation was confirmed in which the tumor growth inhibition rate (TGI) increased in a dose-dependent manner. . When compared to the negative control, the WM-A1-3389 antibody dosage and tumor growth inhibition rate (TGI) are listed in Table 5 below.

	WM-A1-3389 antibody administration group
Dose (mg/kg)	3	10	50
TGI (%)	44.9 ± 3.8	57.0 ± 3.5	66.0 ± 3.5

Example 3. Anticancer activity of WM-A1-3389 antibody against biliary tract cancer

Example 3.1. Confirmation of IGSF1 expression in human biliary tract cancer cell lines

Expression of IGSF1 in Choi-CK cells, a human biliary tract cancer cell line, was confirmed in the same manner as Example 2.1.

As a result, as shown in Figure 8, the proportion of cells expressing IGSF1 in the Choi-CK cell line was found to be about 57.5%.

Example 3.2. Confirmation of changes in cytokine secretion by co-culture of human biliary tract cancer cell lines and peripheral blood cells

Using a co-culture system of biliary tract cancer cell lines and peripheral blood cells, changes in cytokine secretion due to treatment with WM-A1-3389 antibody were confirmed in the same manner as in Example 2.2.

As a result, as shown in Figure 9, the secretion of Granzyme B, as well as cytokines IFN-γ and TNF-α, in Choi-CK biliary tract cancer cells expressing IGSF1 was higher in the group treated with the WM-A1-3389 antibody compared to the control group. A statistically significant increase was confirmed.

Example 3.3. Confirmation of anticancer activity of WM-A1-3389 antibody in xenograft biliary tract cancer cell line transplantation mouse model

The anticancer activity of the WM-A1-3389 antibody was confirmed in a biliary tract cancer mouse model transplanted with a human biliary tract cancer cell line.

Specifically, 6-week-old female peripheral blood mononuclear cell humanized mice (Gem biosciences) were purchased and acclimatized for 1 week, and then Choi-CK cells, a human biliary tract cancer cell line, were diluted in PBS to a concentration of 5Х10 ⁶ cells/mice. It was injected subcutaneously (100 μl) on the dorsal side. Human IgG isotype was used as a negative control for the WM-A1-3389 antibody. When the tumor size reached an average of 80 mm ³ to 100 mm ³ , human IgG isotype (negative control) was administered intraperitoneally at a dose of 10 mg/kg or WM-A1-3389 antibody at a dose of 30 mg/kg. Administration was conducted once every three days for three weeks, and the tumor size and body weight of the mice were measured twice a week. On the 22nd day of administration, the experimental animals were euthanized and the tumors were extracted and weighed.

As a result, as shown in Figure 10, the WM-A1-3389 antibody administration group showed higher tumor growth inhibition efficacy than the negative control group.

When compared to the negative control group, the WM-A1-3389 antibody dosage and tumor growth inhibition rate (TGI) are listed in Table 6 below (***: P<0.001).

	WM-A1-3389 antibody administration group
Dose (mg/kg)	30
TGI (%)	58.0 ± 4.3
P-Value	0.0008 ( *** )

As shown in Table 6, tumor growth inhibition (TGI) was shown to be about 58.0%. In addition, it was confirmed that tumor growth was suppressed in individual subjects.

Example 4. Anticancer activity of WM-A1-3389 antibody against head and neck cancer

Example 4.1. Confirmation of IGSF1 expression in human head and neck cancer cell lines

The expression of IGSF1 in FaDu cells, a human head and neck cancer cell line, was confirmed in the same manner as Example 2.1.

As a result, as shown in Figure 11, the proportion of cells expressing IGSF1 in the FaDu cell line was found to be about 37.7%.

Example 4.2. Confirmation of changes in cytokine secretion by co-culture of human head and neck cancer cell lines and peripheral blood cells

Using a co-culture system of head and neck cancer cell lines and peripheral blood cells, changes in cytokine secretion due to treatment with WM-A1-3389 antibody were confirmed in the same manner as in Example 2.2.

As a result, as shown in Figure 12, the secretion of Granzyme B and cytokines IFN-γ and TNF-α from FaDu head and neck cancer cells expressing IGSF1 was statistically significantly higher in the group treated with the WM-A1-3389 antibody compared to the control group. It was confirmed that there was a significant increase.

Claims (7)

1. A pharmaceutical composition for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer, comprising an anti-IGSF1 antibody or fragment thereof as an active ingredient.
2. According to paragraph 1,

   The anti-IGSF1 antibody has a heavy chain variable region comprising H-CDR1 including the amino acid sequence of SEQ ID NO: 1, H-CDR2 including the amino acid sequence of SEQ ID NO: 2, and H-CDR3 including the amino acid sequence of SEQ ID NO: 3. ; and

   Comprising a light chain variable region comprising L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6, Pharmaceutical composition.
3. According to paragraph 2,

   The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7;

   A pharmaceutical composition wherein the light chain variable region includes the amino acid sequence of SEQ ID NO: 8.
4. According to paragraph 1,

   The anti-IGSF1 antibody is a pharmaceutical composition characterized in that it specifically binds to the C terminus of IGSF1.
5. A method for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer, comprising administering an anti-IGSF1 antibody or fragment thereof to a subject.
6. An anti-IGSF1 antibody or fragment thereof for use in the prevention or treatment of any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer.
7. Use of an anti-IGSF1 antibody or fragment thereof for the manufacture of a medicament for preventing or treating any one cancer selected from the group consisting of colon cancer, biliary tract cancer, and head and neck cancer.

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