WO2023211068A1 - Pharmaceutical composition for cancer treatment, comprising anti-igsf1 antibody and anti-pd-1 antibody - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for cancer prevention or treatment, comprising an anti-IGSF1 antibody and an anti-PD-1 antibody as active ingredients. The anti-IGSF1 antibody, according to the present invention, exhibited high specificity and high binding affinity to IGSF1. The anti-IGSF1 antibody inhibited tumor growth in a humanized mouse transplanted with IGSF1-overexpressing human lung cancer cells, and increased cytokine expression in tumor tissue. In addition, when the anti-IGSF1 antibody and the anti-PD-1 antibody were co-administered into a tumor model mouse transplanted with a mouse colorectal cancer cell line, more excellent tumor growth inhibition activity was exhibited compared to when the anti-IGSF1 antibody or the anti-PD-1 antibody is administered alone. Thus, the pharmaceutical composition comprising the anti-IGSF1 antibody and the anti-PD-1 antibody as active ingredients may be usefully employed for cancer prevention and treatment.

Description

Pharmaceutical composition for cancer treatment comprising anti-IGSF1 antibody and anti-PD-1 antibody

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an anti-IGSF1 antibody or fragment thereof that specifically binds to IGSF1 and an anti-PD-1 antibody as active ingredients.

In the treatment of cancer, chemical treatment methods such as anticancer drugs are effective to some extent, but much research is required due to the various pathogenesis of cancer and resistance to anticancer drugs. Although cancer treatment rates have improved in recent decades due to the development of diagnostic and treatment technologies, the 5-year survival rate for many advanced cancers remains at 5 to 50%. In addition, for some cancers, survival rates have not changed significantly over the past 20 years despite various research and treatments. In particular, cancer cannot be easily treated with conventional cancer treatment regimens, recurrence occurs, and metastasis occurs to other parts, so a more fundamental treatment method is required. Accordingly, there is increasing interest in developing substances to treat cancer by targeting biomarkers that characterize cancer cells, which are believed to be the cause of cancer malignancy, metastasis, and recurrence.

Meanwhile, Republic of Korea Publication No. 2016-0014564 discloses that the IGSF1 (immunoglobulin superfamily member 1) gene can be used as a biomarker for predicting susceptibility to MET (mesenchymal-epithelial transition factor) inhibitors. The above document discloses that an anticancer drug with high therapeutic effect can be selected by determining the sensitivity of each patient using the biomarker before treating the cancer patient.

Recently, immune checkpoint inhibitors such as Keytruda ^® have been in the spotlight. Immune checkpoint inhibitors are anticancer drugs that help activate our body's immune system to attack cancer cells. Anti-PD-1 antibodies, such as Keytruda, bind to a specific receptor (PD-1) on T cells and block the pathway for cancer cells to avoid the surveillance system of activated T cells, allowing T cells in the body to attack cancer cells. By doing so, it exhibits an anti-cancer effect (Korean Publication No. 2018-0030580). Since cancer treatment to date has focused on killing rapidly dividing cells, which is a characteristic of cancer cells, it acts not only on cancer cells but also on rapidly dividing normal cells, causing side effects. However, immunotherapies are known to have few side effects typical of existing anticancer drugs because they use the cancer patient's immune system to affect cancer cells.

Accordingly, the present inventors studied to develop a therapeutic agent to effectively treat cancer and found that when anti-IGSF1 antibody and anti-PD-1 antibody, which specifically binds to the C terminus of IGSF1, are used together, excellent anticancer effect is achieved. The present invention was completed by confirming that it represents.

To achieve the above object, one aspect of the present invention is a pharmaceutical composition for preventing or treating cancer comprising an anti-IGSF1 antibody or fragment thereof that specifically binds to the C terminus of IGSF1 and an anti-PD-1 antibody as active ingredients. provides.

The anti-IGSF1 antibody according to the present invention showed high specificity and high binding affinity to IGSF1. The anti-IGSF1 antibody according to the present invention increased the infiltration of immune cells within the spheroids when IGSF1-overexpressing lung cancer cell spheroids and human peripheral mononuclear cells were co-cultured. In addition, the anti-IGSF1 antibody according to the present invention inhibited tumor growth in humanized mice transplanted with human lung cancer cells overexpressing IGSF1 and increased the expression of cytokines in tumor tissues. Through the above results, it was confirmed that anti-IGSF1 antibody can inhibit tumor growth by increasing the infiltration of immune cells and immune response into lung cancer tissue with increased IGSF1 expression. In addition, when the anti-IGSF1 antibody and anti-PD-1 antibody are administered in combination to tumor model mice transplanted with mouse colon cancer cell lines, the results are superior compared to the single administration of the anti-IGSF1 antibody or anti-PD-1 antibody. It showed tumor growth inhibitory activity. Therefore, a pharmaceutical composition containing anti-IGSF1 antibody and anti-PD-1 antibody as active ingredients can be usefully used for cancer prevention and treatment.

Figure 1 is a diagram confirming the level of IGSF1 expression in IGSF1 overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) and control group (NCI-H292 MOCK) through Western blot and RT-PCR.

Figure 2 shows tumor infiltrating lymphocytes (TILs) present in the spheroids when IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) and control (NCI-H292 MOCK) spheroids were co-cultured with human peripheral mononuclear cells (PBMCs). ) is a drawing confirming this.

Figure 3 shows the distribution of hCD45+ cells using flow cytometry to confirm the presence of tumor-infiltrating lymphocytes in tumor tissues of mice transplanted with IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) or control group (NCI-H292 MOCK). This is a graph showing the results of the analysis.

Figure 4 shows the expression of IGSF1 and the presence of tumor-infiltrating lymphocytes in tumor tissues of mice transplanted with IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) or control group (NCI-H292 MOCK) using immunohistochemistry. This is a drawing showing the results confirmed.

Figure 5 is a graph showing the results of analyzing the binding affinity of the WM-A1-3389 antibody to the IGSF1 antigen using ELISA.

Figure 6 is a graph showing the results of analyzing the binding affinity of the WM-A1-3389 antibody to the intracellular IGSF1 antigen using FACS analysis.

Figure 7 shows the results of analyzing the binding affinity of the WM-A1-3389 antibody to IGSF1 expressed in IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) and control cells (NCI-H292 MOCK) using FACS analysis. This is a graph showing .

Figure 8 shows two types of IGSF1 overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E and HEK293E IGSF1 O/E) and a control (NCI-H292 MOCK and HEK293E MOCK) IGSF1 knockdown (K/D) cell line treated with shIGSF1. This is a graph showing the results of analyzing the binding specificity of the WM-A1-3389 antibody.

Figure 9 shows tumor-infiltrating lymphocytes present in the spheroids after treatment with IgG or WM-A1-3389 antibody when co-culturing IGSF1-overexpressing human lung cancer cell (NCI-H292 IGSF1 O/E) spheroids and human peripheral mononuclear cells (PBMC). This is a diagram confirming (TIL) through a microscope image.

Figure 10 shows co-culture of IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) and control (NCI-H292 MOCK) spheroids with human peripheral mononuclear cells (PBMC), after treatment with IgG or WM-A1-3389 antibody. This is a graph showing the expression of HMGB1 in spheroids.

Figure 11 is a graph showing the tumor size measured in the IgG or WM-A1-3389 antibody administered group in a mouse model transplanted with IGSF1 overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E).

Figure 12 is a graph showing the tumor size for each individual in the IgG or WM-A1-3389 antibody administration group in a mouse model transplanted with IGSF1 overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E).

Figure 13 is a diagram showing the results of analyzing the expression level of IGSF1 in tissues of Caucasian lung cancer patients.

Figure 14 shows tumors in the WM-A1-3389 antibody administration group, the anti-PD-1 antibody administration group, or the WM-A1-3389 antibody and anti-PD-1 antibody combination administration group in a mouse model transplanted with a mouse-derived colon cancer cell line (MC38). This is a graph showing the size measured.

Figure 15 shows tumors in the WM-A1-3389 antibody administration group, anti-PD-1 antibody administration group, or WM-A1-3389 antibody and anti-PD-1 antibody combination administration group in a mouse model transplanted with mouse-derived colon cancer cell line (CT26). This is a graph showing the size measured.

Figure 16 shows the tumor size of the WM-A1-3389 antibody administration group, the anti-PD-1 antibody administration group, or the WM-A1-3389 antibody and anti-PD-1 antibody combination administration group in a mouse model transplanted with a mouse-derived lung cancer cell line (LLC1). This is a graph shown by measuring .

One aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an anti-IGSF1 antibody or fragment thereof that specifically binds to the C terminus of IGSF1 and an anti-PD-1 antibody as active ingredients.

anti-PD-1 antibody

As used herein, the term “PD-1 (programmed cell death protein 1)” refers to CD279, and is a protein expressed on the surface of activated T cells. It reacts with PD-L1 (B7-H1) and PD-L2 (B7-DC), proteins on the surface of cancer cells, and inhibits T-cell activation, growth factors, and cytokine production mediated by TCR (T cell receptor) and CD28. This induces voice signal transmission. PD-1 inhibitors include, for example, pembrolizumab (Keytruda ^® ), MK-3475, nivolumab (Opdivo ^® ), cemiplimab (Liptayo ^® ), JTX-4014, spartalizumab, Camreli Zumab, sintilimab, thyslerizumab, toripalimab, dostalimab, INCMGA00012, AMP-224, and AMP-514.

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 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.

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 may include a heavy chain variable region (VH) comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, and H-CDR3 of SEQ ID NO: 3. In addition, the IGSF1-specific antibody and fragment thereof of the present invention may include a light chain variable region (VL) comprising L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6. there is. At this time, the heavy chain variable region may have the amino acid sequence of SEQ ID NO: 7, and the light chain variable region may have 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 is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or It may comprise or consist of an amino acid sequence having about 99% identity or 100% identity. In addition, the light chain variable region of the antibody is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% of the amino acid sequence of SEQ ID NO: 8. % or about 99% identity or 100% identity.

Immunoglobulin heavy chain constant regions (CH) exhibit different amino acid compositions and sequences and therefore possess different types of antigenicity. Therefore, 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 a scFv fragment, which is an Fv fragment that binds to IGSF1, as well as a Fab fragment, Fab' fragment, and F(ab')2 fragment with antigen-binding activity, and 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 and the anti-PD-1 antibody of the present invention as active ingredients exhibits preventive or therapeutic efficacy against cancer.

At this time, the cancer may be a cancer in which IGSF1 is overexpressed.

In addition, the above cancers include stomach cancer, liver cancer, lung cancer, non-small cell lung cancer, colon cancer, bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, larynx cancer, esophagus cancer, pancreas cancer, and tongue cancer. , it may be any one selected from the group consisting of skin cancer, sinus tumor, uterine cancer, head cancer, cervical cancer, gallbladder cancer, oral cancer, anal cancer, colon cancer, and central nervous system tumor.

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

In the pharmaceutical composition for preventing or treating cancer of the present invention, the anti-IGSF1 antibody or fragment thereof and the anti-PD-1 antibody are used in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as they can exhibit anticancer activity. may be included. Here, “effective amount” refers to the amount of an 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. can be decided. 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 can 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 may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerogelatin, 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 antibody or composition of the present invention can be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

The term "administration" used herein 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 “pharmacologically 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. It 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, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, treatment period, drugs combined or used simultaneously, 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 that is effective in treating cancer.

Specifically, the effective amount of antibody in the composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally about 0.1 mg to about 1,000 mg, or about 5 mg to about 200 mg per kg of body weight daily. It can be administered every other day or every 2 to 3 weeks, or it can be administered once to three 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 “individual” refers to an object to which the composition of the present invention can be applied (prescribed), and may be a mammal such as a rat, mouse, or livestock, including humans. Preferably, it may be a human, but is not limited thereto.

The antibody of the present invention or a pharmaceutical composition containing the same 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 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 provides the use of a pharmaceutical composition comprising the anti-IGSF1 antibody or fragment thereof and the anti-PD-1 antibody of the present invention as active ingredients for preparing a drug for preventing or treating cancer. Here, anti-IGSF1 antibody and fragment thereof, anti-PD-1 antibody, cancer, prevention and treatment are the same as described above.

Another aspect of the present invention provides a use for cancer prevention or treatment of a pharmaceutical composition comprising the anti-IGSF1 antibody or fragment thereof and the anti-PD-1 antibody of the present invention as active ingredients. Here, anti-IGSF1 antibody and fragment thereof, anti-PD-1 antibody, cancer, prevention and treatment are the same as described above.

Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition containing an anti-IGSF1 antibody or fragment thereof and an anti-PD-1 antibody as active ingredients. Here, anti-IGSF1 antibody and fragment thereof, anti-PD-1 antibody, 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 cancer patient or an individual at a high risk of suffering from cancer.

The administration route, dosage, and frequency of administration of the pharmaceutical composition may be administered to the subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency of administration may 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 cancer, or may be formulated in the form of a combination preparation with other drugs.

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 through PCR from the Jurkat cell cDNA library, a human Fc (fragment crystallizable region) and a His-tag were attached to the carboxy terminus (C terminus) using the N293F vector (Y Biologics Co., Ltd.). (His-tag) was fused to create an IGSF1 protein expression vector. 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). Afterwards, the IGSF1 extracellular domain was first purified using protein A agarose, and then the IGSF1 extracellular domain was secondarily purified using Superdex 200 gel filtration chromatography, and then used for antibody selection.

Example 1.2. Selection of IGSF1 human antibodies

After coating and blocking the IGSF1 antigen, biopanning (Y Biologics Co., Ltd.) was performed using the prepared human antibody library phage (Y Biologics Co., Ltd.) to elute only phages that specifically bound to the antigen. did. 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 with high binding capacity were selected from the third round of panning, and preliminary antibody clones were secured 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 through this confirmation were converted from phage to whole IgG vectors. It was confirmed that the heavy chain 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.

WM-A1-3389
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 (heavy chain)	QVQLVQSGAEVKRPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGRIIPFVGTVDYAQKFQDRVTITADKSTNTAYMELSSLRSEDTAVYYCVRDGGRSYFDSWGPGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	21
light chain (light chain)	QFVLTQPPSVSAAPGQDVIISCSGNTSNIGSNLVSWFQQFPETAPKLLIYDNHKRPSGISDRFSGTKSGTSASLAISGLQSEDEADYYCVAWDDSLNGYVFGTGTKVTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC	22

Example 2. Analysis of the relationship between IGSF1 expression and tumor infiltrating lymphocytes

Example 2.1. Construction of IGSF1 overexpressing cell lines

IGSF1 was overexpressed in NCI-H292, a human lung cancer cell line, or HEK293E, a human embryonic kidney cell line, to create a cell line in which IGSF1 was overexpressed (Figure 1). At this time, MOCK is a control group without IGSF1 expression.

Specifically, an expression vector containing a polynucleotide encoding IGSF1 (OriGene Technologies, Inc., Cat No.RC209621) was transfected into human lung cancer cell line NCI-H292 cells or human embryonic kidney cell line HEK293F cells. Afterwards, the transfected cells were selected by culturing them in a medium containing G418 (neomycin). The IGSF1 expression level was confirmed for the selected clones, and the clone showing the highest IGSF1 expression was selected and used in the experiment. MOCK refers to an empty vector in which the polynucleotide encoding IGSF1 is not loaded.

Example 2.2. Analysis of the association between IGSF1 expression and tumor-infiltrating lymphocytes in lung cancer cell line spheroids

To confirm the correlation between IGSF1 expression and tumor-infiltrating lymphocytes (TIL) in lung cancer at the cellular level, tumor-infiltrating lymphocytes were identified in spheroids using lung cancer cells that overexpressed IGSF1.

First, to produce lung cancer cell spheroids, NCI-H292 IGSF1 O/E cells and NCI-H292 MOCK cells were each plated at 2×10 ⁴ cells/well in a U-Bottom 96-well plate (Nunc, 174925). ) were seeded and cultured in a CO ₂ incubator at 37°C for 72 hours. Peripheral blood mononuclear cells (PBMC) were prepared by centrifuging at 1,200 rpm for 10 minutes to remove the supernatant and resuspending in PBS. 18 ㎕ of DMSO was added to CFSE (carboxyfluorescein succinimidyl ester, Invitrogen, C34554) to make a concentration of 5 mM, and then diluted to 1 mM in PBS. 1 μl of 1 mM CFSE solution was added per 1 × 10 ⁶ cells/ml of prepared peripheral blood mononuclear cells, and then stained for 10 minutes in a CO ₂ incubator at 37°C. Afterwards, medium containing FBS (fetal bovine serum) in an amount five times the amount of PBS was added to the solution containing stained peripheral blood mononuclear cells (PBMC). Afterwards, the reaction was performed at 37°C in a CO ₂ incubator for 5 minutes. After removing the supernatant by centrifugation at 1,200 rpm for 10 minutes, the stained peripheral blood mononuclear cells were resuspended in medium to prepare peripheral blood mononuclear cells to be used in the experiment.

Afterwards, the formed spheroids were transferred to an ultra-low attachment 96-well plate (Corning, CLS3474), 2 wells at a time, and CFSE-stained peripheral blood mononuclear cells were inoculated at 1×10 ⁵ cells/well and incubated at 37°C in a CO ₂ incubator. were co-cultured for 24 hours. Tumor-infiltrating lymphocytes (TIL) were observed through fluorescence microscopy. At this time, NCI-H292 MOCK cells were used as a control for IGSF1 overexpressing cells.

As a result, it was confirmed that tumor infiltrating lymphocytes (TIL) were reduced in IGSF1-overexpressing human lung cancer cell (NCI-H292 IGSF1 O/E) spheroids compared to the control group (NCI-H292 MOCK) (Figure 2).

Example 2.3. Analysis of the relationship between IGSF1 expression and tumor-infiltrating lymphocytes in tumor tissues of humanized mice transplanted with lung cancer cell lines.

To confirm the correlation between the expression of IGSF1 and tumor infiltrating lymphocytes (TIL) in lung cancer at the in vivo level, tumor infiltrating lymphocytes were identified in the tumor tissues of humanized mice transplanted with human lung cancer cells that overexpressed IGSF1.

Specifically, NSG mice (SID (NSGA) mice, F) transplanted with human peripheral blood mononuclear cells (PBMC) were treated with NCI-H292 IGSF1 O/E cells, a human lung cancer cell line that overexpresses IGSF1, or NCI-H292 MOCK cells as a control group. Tumor parts of mice were collected 17 days after transplantation with 5×10 ⁶ cells per mouse.

Human peripheral blood mononuclear cells were isolated from each collected tumor, analyzed by FACS, and the level of tumor infiltrating lymphocytes and IGSF1 expression within the tumor tissue was confirmed through immunohistochemistry. To identify human peripheral blood mononuclear cells infiltrating the tumor, the tumor tissue was first treated with collagenase B (Roche, cat. #11088815001) and reacted at 37°C for more than 2 hours to decompose the tumor tissue. When the tumor tissue was completely decomposed into cells, it was separated into single cells by pipetting with a 1 ml pipette.

The isolated single cells were transferred to a 50 ml tube (SPL, cat. #50050) and washed with 20 ml of PBS. Afterwards, it was centrifuged at 1,200 rpm for 3 minutes and the supernatant was removed. The remaining cells were treated with DNase I (Roche, cat. #11284932001) and incubated at 37°C for 20 minutes. After the reaction, 20 ml of PBS was added, and the supernatant was removed by centrifugation at 1,200 rpm for 3 minutes. The remaining cells were treated with 0.25% Trysin/EDTA (GIBCO, cat. #15400-054). After mixing the cells well, a cell strainer (SPL, cat. #93070) was placed on a new 50 ml tube and the cells were filtered. 20 mL of PBS was added to the tube containing the filtered cells and mixed well. Afterwards, centrifugation was performed at 1,200 rpm for 3 minutes and the supernatant was removed. 1 ml of Stain Buffer (BD, cat. #554656) was added to the remaining cells and washed.

To block the non-specific antibody response of the isolated cells, 2 μg of Human Fc block (BD, cat. #564219) was added and reacted at room temperature for 10 minutes. After reaction, anti-human CD45 (BD, cat. #564357) antibody was added and reaction was performed in a dark state at 4°C for 30 minutes. After reaction, 1 ml of Stain Buffer was added and washed. Afterwards, the cells were collected by centrifuging at 1,200 rpm for 3 minutes and removing the supernatant. 200 ㎕ of Stain Buffer was added to the collected cells and analyzed with a BD LSRFortessa ^TM Flow Cytometer (Figure 3).

In addition, the distribution of tumor lymphocytes and the expression of IGSF1 expressed within the tumor were confirmed through immunohistochemical staining. Specifically, NSG mice (SID (NSGA) mice, F) transplanted with human peripheral blood mononuclear cells (PBMC) were injected with 5 Transplanted with ⁶ cells. Sections of tumor tissue collected from mice 17 days after transplantation were deparaffinized and rehydrated.

Afterwards, for heat-induced epitope recovery, the cells were immersed in target recovery buffer and heated in a microwave oven for 15 minutes. After heating, the cells were left in target recovery buffer for 30 minutes, washed three times with Tris-buffered saline-0.05% Tween 20 (TBS-T), and blocked with blocking solution for 60 minutes. As the primary antibody, anti-IGSF1 antibody (Santacruz, sc-393786) was diluted 1:100 and allowed to bind overnight at 4°C. The next day, it was washed three times with TBS-T and reacted with endogenous peroxidase blocking reagent (Cell Marque, 925B) at room temperature for 5 minutes. Afterwards, secondary antibodies (Vector, PK-6101 PK-6102) were bound at room temperature for 60 minutes. After washing three times with TBS-T, it was reacted with avidin-biotin for 60 minutes. After final DAB staining (Vector, SK-4100), tissue staining was completed through a dehydration process, and the stained tissue sections were observed under a microscope.

As a result, it was confirmed that hCD45+ cells, a human immune cell, decreased in the tumor tissues of humanized mice transplanted with IGSF1-overexpressing human lung cancer cells (NCI-H292 IGSF1 O/E) compared to the control group (NCI-H292 MOCK) (Figure 4 ).

Example 3. Binding affinity analysis of anti-IGSF1 antibody

Example 3.1. In vitro Binding affinity analysis of anti-IGSF1 antibodies at the level

The binding affinity of the WM-A1-3389 antibody to the IGSF1 antigen was confirmed at the in vitro level using ELISA analysis.

Specifically, 100 ng IGSF1 was treated to coat a 96-well plate, and then 200 μl of 4% skim milk (PBST) was added and blocked for 1 hour at room temperature. The WM-A1-3389 antibody was serially diluted in 4% skim milk (PBST) at 12 concentrations of 1/3 each and then reacted at room temperature for 2 hours. After the reaction was completed, the wells were washed with PBST, treated with human IgG Fc-HRP antibody, and reacted at room temperature for 1 hour. Next, the wells were washed with PBST, and TMB peroxidase substrate was added to check the degree of color development. At this time, for confirmation, the absorbance was measured at a wavelength of 450 nm and the results were compared and analyzed.

As a result of the analysis, the Kd value of the WM-A1-3389 antibody was 2.2×10 ^-11 , showing high binding affinity to the IGSF1 antigen (FIG. 5).

Example 3.2. Binding affinity analysis of anti-IGSF1 antibodies to intracellular IGSF1

To confirm the binding affinity of the WM-A1-3389 antibody to the IGSF1 antigen at the cellular level, human lung cancer cells overexpressing IGSF1 (NCI-H292 IGSF1 O/E) and a control group (NCI-H292 MOCK) were used to determine the binding affinity of the WM-A1-3389 antibody to the IGSF1 antigen. The binding ability of the WM-A1-3389 antibody was confirmed.

Specifically, the medium of NCI-H292 IGSF1 O/E cells and NCI-H292 MOCK cells was removed, washed once with PBS, and then treated with 2 ml of 0.25% trypsin-EDTA to separate the cells. The separated cells were diluted with 8 ml of PBS (hereinafter referred to as FACS buffer) containing 2% FBS and 0.05% sodium azide, and then centrifuged at 1,200 rpm for 1 minute to remove the supernatant. Afterwards, the cells were resuspended in FACS buffer to 1×10 ⁵ cells/ml. After resuspension, cells were dispensed in 1 ml portions into FACS tubes and centrifuged at 1,200 rpm for 1 minute to remove the supernatant.

The cell pellet remaining in the FACS tube was vortexed, and WM-A1-3389 antibody was diluted by 1/4 from 20 μM to 0 μM per 200 μl of FACS buffer and added at a total of 12 concentrations. , and reacted at 4°C for 30 minutes. 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. This process was performed a total of two times. Additionally, the cell pellet remaining in the FACS tube was vortexed, and FITC-labeled goat anti-human IgG antibody (Invitrogen, 62-8411) was added at 5 ㎍/㎖ per 200 ㎕ of FACS buffer and incubated at 4°C for 30 minutes. Reacted for minutes. After completion of the reaction, 1 ml of FACS buffer was added to each tube and centrifuged at 1,200 rpm for 1 minute to remove the supernatant. This process was performed a total of two times.

Finally, after removal of the supernatant, the remaining cell pellet was resuspended in 200 μl of FACS buffer and subjected to FACS analysis. FACS analysis measured the fluorescence value of FITC labeled in each cell using a BD LSRFortessa ^TM Flow Cytometer. Afterwards, the results were analyzed using FlowJo software, and the EC50 value was calculated using the sigma plot program. At this time, NCI-H292 MOCK cells were used as a control for IGSF1 overexpressing cells.

As a result of the analysis, no binding was confirmed in the control group (NCI-H292 MOCK) regardless of the concentration of WA-A1-3389 antibody. On the other hand, in human lung cancer cells overexpressing IGSF1 (NCI-H292 IGSF1 O/E), the EC50 value of the WM-A1-3389 antibody was confirmed to be 69 nM (FIG. 6).

Example 4. Analysis of antigen specificity of anti-IGSF1 antibody against intracellular IGSF1 antigen

In order to analyze the antigen-specific binding ability (target selectivity) of the WM-A1-3389 antibody to the IGSF1 antigen at the cellular level, human lung cancer cells overexpressing IGSF1 (NCI-H292 IGSF1 O/E) were used. The degree of binding of the WM-A1-3389 antibody to expressed IGSF1 was confirmed.

Specifically, the medium of human lung cancer cells overexpressing IGSF1 (NCI-H292 IGSF1 O/E) and its control group (NCI-H292 MOCK) was removed, washed once with PBS, and then added with 2 ml of 0.25% trypsin-EDTA. Cells were separated by treatment. The separated cells were diluted with 8 ml of PBS (hereinafter referred to as FACS buffer) containing 2% FBS and 0.05% sodium azide, and then centrifuged at 1,200 rpm for 1 minute to remove the supernatant. Afterwards, the cells were resuspended in FACS buffer to 1×10 ⁵ cells/ml. After resuspension, cells were dispensed in 1 ml portions into FACS tubes and centrifuged at 1,200 rpm for 1 minute to remove the supernatant.

The cell pellet remaining in the FACS tube was vortexed, and 0.4 μg of human IgG isotype antibody (Bio After completion of the reaction, 1 ml of FACS buffer was added to each tube and centrifuged at 1,200 rpm for 1 minute to remove the supernatant. This process was performed a total of two times. The cell pellet remaining in the FACS tube was vortexed, and 0.4 μg of FITC-labeled goat anti-human IgG antibody (Invitrogen, 62-8411) was added per 200 μl of FACS buffer and reacted 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. This process was performed a total of two times. Finally, after removal of the supernatant, the remaining cell pellet was resuspended in 200 μl of FACS buffer and subjected to FACS analysis. For FACS analysis, the fluorescence value of FITC labeled in each cell was measured using a BD LSRFortessa ^TM Flow Cytometer, and the results were analyzed using FlowJo software. At this time, NCI-H292 MOCK cells were used as a control for IGSF1 overexpressing cells, and human IgG isotype was used as a control for WM-A1-3389 antibody.

As a result, the WM-A1-3389 antibody treatment group showed a binding affinity of 2.6% in the control group (NCI-H292 MOCK) and 78.9% in IGSF1 overexpressing cells (NCI-H292 IGSF1 O/E) compared to the IgG isotype treatment group. Binding was shown (Figure 7).

Next, shRNA (hereinafter referred to as shIGSF1) that specifically binds to the mRNA encoding IGSF1 was transfected into NCI-H292 IGSF1 O/E cells and HEK293E IGSF1 O/E cells in which IGSF1 was overexpressed, thereby reducing the expression of IGSF1. (hereinafter referred to as IGSF1 K/D cells), the binding affinity of the WM-A1-3389 antibody to the intracellular IGSF1 antigen was measured. At this time, scramble RNA (hereinafter referred to as sc cells) without shIGSF1 was used as a control for transfection (IGSF1 K/D), and human IgG isotype was used as a control for the WM-A1-3389 antibody. The antigen specificity of the WM-A1-3389 antibody was compared with its binding affinity to IGSF1 K/D cells based on its binding affinity to sc cells. Additionally, NCI-H292 MOCK cells and HEK293E MOCK cells were used as controls for IGSF1 overexpressing cells, respectively.

Specifically, the medium of NCI-H292 (IGSF1 O/E and MOCK) and HEK293E (IGSF1 O/E and MOCK) cell lines was removed, washed once with PBS, and NCI-H292 cells were incubated with 0.25% trypsin-EDTA, HEK293E cells were separated by treating each cell with 2 ml of 0.05% trypsin-EDTA. The separated cells were diluted with 8 ml of culture medium and then centrifuged at 800 rpm for 3 minutes to remove the supernatant. The remaining cells were resuspended to a concentration of 1×10 ⁵ cells/ml (NCI-H292) and 0.5×10 ⁵ cells/ml (HEK293E), respectively, and then added at 3 ml each to a 60 mm culture plate and incubated at 37°C. It was cultured in an incubator for one day. shIGSF1 transfection was performed the next day. 200 ㎕ of jet PRIME buffer and 10 nM of shIGSF1 were added to a 1.5 ㎖ tube and mixed. Afterwards, 4 ㎕ of jet PRIME reagent was added, mixed, and reacted at room temperature for 10 minutes. Additionally, after replacing the cell medium prepared the day before, 200 ㎕ of the transfection mixture was added to each cell and reacted in a cell incubator for 24 hours. After 24 hours, it was replaced with new culture medium and cultured for an additional 24 hours.

The medium was removed from the transfected cells, and FACS analysis was performed in the same manner as above.

As a result, WM-A1-3389 antibody binding was confirmed in the sc cell line compared to the human IgG isotype treatment group. In addition, when IGSF1 expression was reduced based on the binding affinity (IGSF1 K/D cells), it was confirmed that the binding affinity of the WM-A1-3389 antibody also decreased (Figure 8).

Example 5. Analysis of immune anti-cancer efficacy of anti-IGSF1 antibody in lung cancer cell spheroids

To analyze the immuno-anticancer efficacy of the WM-A1-3389 antibody at the cellular level, lung cancer cell spheroids and peripheral blood mononuclear cells (PBMC) were co-cultured to determine tumor-infiltrating lymphocytes (TIL) and immunogenic cell death.

Co-culture of lung cancer cell spheroids and peripheral blood mononuclear cells was performed in the same manner as Example 2.2.

The co-cultured cells and supernatant were collected in a tube and centrifuged at 1,200 rpm for 2 minutes to remove the supernatant. The cell pellet was treated with 500 ㎕ of 0.25% trypsin-EDTA to form single cells, and then diluted with 2 ㎖ of PBS (hereinafter referred to as FACS buffer) to which 2% FBS and 0.05% NaN ₃ were added. Afterwards, the supernatant was removed by centrifugation at 1,200 rpm for 3 minutes. The remaining cell pellet was resuspended in 200 ㎕ of FACS buffer, and anti-HMGB1 antibody (Biolegend, 651408) was added and stained at 4°C for 30 minutes.

1 ml of FACS buffer was added to each tube, centrifuged at 1,200 rpm for 2 minutes, and the supernatant was removed. This process was performed a total of two times. Afterwards, it was analyzed using a BD LSRFortessa ^TM Flow Cytometer. FACS analysis results were analyzed using FlowJo software. Additionally, tumor infiltrating lymphocytes (TIL) were observed through fluorescence microscopy. At this time, human IgG isotype was used as a control for the WM-A1-3389 antibody.

As a result, it was confirmed that tumor infiltrating lymphocytes (TIL) increased in the WM-A1-3389 antibody treatment group in IGSF1 overexpressing lung cancer cell (NCI-H292 IGSF1 O/E) spheroids compared to the control group (FIG. 9). In addition, immunogenic cell death (ICD) was confirmed to increase in the WM-A1-3389 antibody treatment group compared to the control group in IGSF1-overexpressing lung cancer cell spheroids (FIG. 10).

Example 6. By administering anti-IGSF1 antibody alone in a tumor mouse model transplanted with a lung cancer cell line Tumor growth inhibition efficacy analysis

To confirm the anticancer efficacy of the WM-A1-3389 antibody at the animal level, human lung cancer cells overexpressing IGSF1 (NCI-H292 IGSF1 O/E) were transplanted into peripheral blood mononuclear cell humanized model (PBMC humanized model) mice. Then, the tumor growth inhibition efficacy of the WM-A1-3389 antibody was evaluated.

Specifically, 6-week-old female peripheral blood mononuclear cell humanized mice (Gem biosciences) were purchased and acclimatized for 1 week, and then IGSF1-overexpressing human lung cancer cells NCI-H292 IGSF1 O/E (5 × 10 ⁶ cells/mouse) were added to PBS. and was diluted in Matrigel and injected subcutaneously (200 ㎕) into the right dorsal side of the mouse. When the tumor size reached approximately 120 mm ³ , IgG isotype (control group) or WM-A1-3389 antibody was administered intraperitoneally at a dose of 10 mg/kg, respectively. Administration was performed once every 3 days for 4 weeks, and the tumor size and body weight of the mice were measured twice a week. On the 22nd day of administration, blood and tumors from satellite group mice were collected and FACS analysis was performed. After administration was completed, the experimental animals were euthanized, the tumors were extracted, and their weight was measured. Human IgG isotype was used as a control for the WM-A1-3389 antibody.

As a result, the WM-A1-3389 antibody administration group showed higher tumor growth inhibition efficacy than the control group, and showed a tumor inhibition rate (TGI) of about 64.5% (FIG. 11). In addition, it was confirmed that tumor growth was suppressed in individual subjects (FIG. 12).

Example 7. Analysis of IGSF1 expression in lung cancer patient tissues of Caucasian ethnicity

The expression of IGSF1 in tissues of Caucasian lung cancer patients was confirmed by immunohistochemical staining.

Specifically, human non-small cell lung cancer patient tissue sections were deparaffinized and rehydrated. Thereafter, for heat-induced epitope recovery, it was immersed in target recovery buffer and heated in a microwave oven for 15 minutes. After heating, it was further reacted in target recovery buffer for 30 minutes. Afterwards, it was washed three times with Tris-buffered saline 0.05% Tween 20 (TBS-T), and then blocked with blocking solution for 60 minutes. As the primary antibody, anti-IGSF1 antibody (Santacruz, sc-393786) was diluted 1:100 and allowed to bind overnight at 4°C. The next day, the tissue sections were washed three times with TBS-T and then reacted with endogenous peroxidase blocking reagent (Cell Marque, 925B) for 5 minutes. Afterwards, it was treated with secondary antibody (Vector, PK-6101 PK-6102) and allowed to bind at room temperature for 60 minutes. After binding, the mixture was washed three times with TBS-T. After washing, avidin-biotin was treated and reacted for 60 minutes. Afterwards, DAB staining (Vector, SK-4100) was performed, and the stained tissue sections were observed through a microscope (FIG. 13).

Example 8. Analysis of tumor growth inhibition efficacy by combined administration of anti-IGSF1 antibody and anticancer agent in tumor mouse model transplanted with various cancer cell lines

Example 8.1. Antitumor effect of combined administration of anti-IGSF1 antibody and anticancer agent on syngeneic mouse model transplanted with colon cancer cell line MC38

To confirm the anticancer efficacy of combined administration of WM-A1-3389 antibody and anti-PD-1 antibody at the animal level, WM-A1-3389 antibody and anti-PD-1 were administered to a mouse model transplanted with MC38, a colon cancer cell line. After co-administration of the antibodies, the tumor growth inhibition efficacy was evaluated.

Specifically, 5-week-old female C57BL/6 mice (Orient Bio) were purchased and acclimatized for 1 week, and then the mouse colon cancer cell line MC38 (2.5 × 10 ⁵ cells/mouse) was diluted in PBS and injected subcutaneously on the right ventral side of the mouse. (100 μl) was injected. When the tumor size reached approximately 100 mm ³ , mIgG (negative control) (Sigma Aldrich, I5381), WM-A1-3389 antibody, or anti-PD-1 antibody (Bio Each was administered intraperitoneally at a dose of 10 mg/kg, or WM-A1-3389 antibody and anti-PD-1 antibody were each administered at a dose of 10 mg/kg, for a total of 20 mg/kg. Administration was conducted once every three days for two weeks, and the tumor size and body weight of the mice were measured twice a week. After administration was completed, the experimental animals were euthanized, the tumors were extracted, and their weight was measured.

As a result, compared to the negative control group, tumor growth inhibition efficacy was confirmed in all the WM-A1-3389 antibody administration group, anti-PD-1 antibody administration group, and WM-A1-3389 antibody and anti-PD-1 antibody combination administration group. In particular, the group administered with the WM-A1-3389 antibody and anti-PD-1 antibody showed superior tumor growth inhibition efficacy compared to the group administered with the WM-A1-3389 antibody or anti-PD-1 antibody alone (FIG. 14 and Table 3).

	WM-A1-3389 antibody	anti-PD-1 antibody	Combined use (WM-A1-3389 antibody + anti-PD-1 antibody)
Dosage administered (mg/kg)	10	10	10 + 10
TGI (%)	39.1 ± 7.2	43.3 ± 5.2	68.2 ± 8.1

Example 8.2. Anti-tumor effect following combined administration of anti-IGSF1 antibody and anticancer agent on syngeneic mouse model transplanted with colon cancer cell line CT26

In order to confirm the anticancer efficacy of the combined administration of the WM-A1-3389 antibody and the anti-PD-1 antibody, the same method as Example 8.1 was performed, except that in this example, CT26, a colon cancer cell line, was used as the transplant cell line. And, 5-week-old female BALB/c mice were used as recipient mice.

As a result of analyzing the efficacy of tumor growth inhibition after co-administration of WM-A1-3389 antibody and anti-PD-1 antibody to a mouse model transplanted with CT26, a colon cancer cell line, the WM-A1-3389 antibody group was compared to the negative control group. , the tumor growth inhibition efficacy was confirmed in both the anti-PD-1 antibody administration group and the WM-A1-3389 antibody and anti-PD-1 antibody combination administration group. In particular, the group administered with the WM-A1-3389 antibody and anti-PD-1 antibody showed superior tumor growth inhibition efficacy compared to the group administered with the WM-A1-3389 antibody or anti-PD-1 antibody alone (FIG. 15 and Table 4).

	WM-A1-3389 antibody	anti-PD-1 antibody	Combined use (WM-A1-3389 antibody + anti-PD-1 antibody)
Dosage administered (mg/kg)	10	10	10 + 10
TGI (%)	39.8 ± 5.6	3.6 ± 8.6	73.3 ± 5.8

Example 8.3. Antitumor effect of combined administration of anti-IGSF1 antibody and anticancer drug on syngeneic mouse model transplanted with lung cancer cell line LLC1

In order to confirm the anticancer efficacy of the combined administration of the WM-A1-3389 antibody and the anti-PD-1 antibody, the same method as Example 8.1 was performed, except that in this example, the lung cancer cell line LLC1 was used as the transplant cell line. , Administration was conducted once every three days for 11 days.

When WM-A1-3389 antibody and anti-PD-1 antibody were co-administered to a mouse model transplanted with LLC1, a lung cancer cell line, and the tumor growth inhibition efficacy was analyzed, compared to the negative control group, the WM-A1-3389 antibody administration group, Tumor growth inhibition efficacy was confirmed in both the anti-PD-1 antibody administration group and the WM-A1-3389 antibody and anti-PD-1 antibody combination administration group. In particular, the group administered with the WM-A1-3389 antibody and anti-PD-1 antibody showed superior tumor growth inhibition efficacy compared to the group administered with the WM-A1-3389 antibody or anti-PD-1 antibody alone (FIG. 16 and Table 5).

	WM-A1-3389 antibody	anti-PD-1 antibody	Combined use (WM-A1-3389 antibody + anti-PD-1 antibody)
Dosage administered (mg/kg)	10	10	10 + 10
TGI (%)	55.4 ± 10.5	22.2 ± 12.0	84.9 ± 2.6

Claims (5)

1. A pharmaceutical composition for preventing or treating cancer comprising an anti-IGSF1 antibody or fragment thereof that specifically binds to the C terminus of IGSF1 and an anti-PD-1 antibody as active ingredients.
2. According to paragraph 1,

   The anti-IGSF1 antibody includes a heavy chain variable region comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, and H-CDR3 of SEQ ID NO: 3; and

   A pharmaceutical composition for preventing or treating cancer, comprising a light chain variable region including L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6.
3. According to paragraph 2,

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

   A pharmaceutical composition for preventing or treating cancer, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 8.
4. According to paragraph 1,

   The anti-PD-1 antibodies include pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, thyslerizumab, toripalimab, dostalimab, INCMGA00012 , a pharmaceutical composition for preventing or treating cancer, which is any one selected from the group consisting of AMP-224 and AMP-514.
5. According to paragraph 1,

   The above cancers include stomach cancer, liver cancer, lung cancer, non-small cell lung cancer, colon cancer, ductal cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, larynx cancer, esophagus cancer, pancreatic cancer, tongue cancer, and skin cancer. A pharmaceutical composition for preventing or treating cancer, which is any one selected from the group consisting of sinus cancer, uterine cancer, head cancer, cervical cancer, gallbladder cancer, oral cancer, anal cancer, colon cancer, and central nervous system tumor.

Images