Mouse SCF-neutralizing antibody and uses thereof

Abstract

The present invention relates to a mouse SCF-neutralizing antibody and uses thereof. Specifically, the present invention relates to an antibody for neutralizing mouse SCF, comprising a heavy and a light chain of specific sequences, or an antigen-binding fragment thereof. A novel antibody is constructed for neutralizing SCF to inhibit SCF / c-kit signaling, leading to the development of a therapeutic agent for inhibiting abnormal angiogenesis, suppressing differentiation, proliferation, and degranulation of mast cells, and restraining proliferation of smooth muscle cells.

Description

Antibody neutralizing mouse SCF and its uses

[0001] The present invention relates to an antibody that neutralizes mouse stem cell factor (SCF) and the use thereof.

[0002] Stem Cell Factor (SCF) / c-kit is known to play a major role in cell differentiation and proliferation, as well as in various pathological conditions.

[0003] SCF signals are transmitted into the cell after binding to tyrosine kinase-type C-kit receptors. Although the effect of SCF alone is weak, it exhibits significant synergistic effects with many cytokines, such as IL-I, IL-3, IL-4, IL-6, IL-7, IL-11, G-CSF, GM-CSF, EPO, and IFM-γ, and sustains the proliferation of hematopoietic stem cells, various hematopoietic progenitor cells, and mast cells. In vitro administration demonstrates an increase in hematopoietic stem cells or systemic mast cells.

[0004] Meanwhile, the current situation is that antibodies that specifically or effectively bind to these SCF proteins have not been developed.

[0005] The object of the present invention is to provide an antibody or an antigen-binding fragment thereof that neutralizes mouse stem cell factor (SCF).

[0006] Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or its antigen-binding fragment, a recombinant expression vector comprising the nucleic acid molecule, and a cell isolated by transforming with the recombinant expression vector.

[0007] Another objective of the present invention is to provide a hybridoma cell line that produces the antibody or its antigen-binding fragment.

[0008] Another objective of the present invention is to provide a composition for detecting SCF antigens comprising the antibody or its antigen-binding fragment as an active ingredient.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of pulmonary arterial hypertension comprising the antibody or its antigen-binding fragment as an active ingredient.

[0010] To achieve the above objective, the present invention provides a 2E7 antibody or an antigen-binding fragment thereof that neutralizes a mouse stem cell factor (SCF), comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 1, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 2, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 3; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 4, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 5, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 6.

[0011] In addition, the present invention provides a 4B11 antibody or its antigen-binding fragment for neutralizing mouse SCF, comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 15, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 16, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 17; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 18, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 19, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 20.

[0012] In addition, the present invention provides a 4E2 antibody for neutralizing SCF or an antigen-binding fragment thereof comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 29, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 30, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 31; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 32, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 33, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 34.

[0013] In addition, the present invention provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0014] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0015] In addition, the present invention provides a cell isolated by transforming with the recombinant expression vector.

[0016] In addition, the present invention provides a hybridoma cell line that produces the antibody or its antigen-binding fragment.

[0017] In addition, the present invention provides a composition for detecting SCF antigens comprising the antibody or its antigen-binding fragment as an active ingredient.

[0018] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of pulmonary arterial hypertension comprising the antibody or its antigen-binding fragment as an active ingredient.

[0019] The present invention relates to an antibody that neutralizes mouse SCF and its uses. Specifically, the present invention relates to an antibody that neutralizes mouse SCF comprising heavy and light chains of specific sequences, or to an antigen-binding fragment thereof. By producing a novel antibody that neutralizes SCF and inhibiting SCF / c-kit signaling, it is possible to develop a therapeutic agent that inhibits abnormal angiogenesis, inhibits the differentiation, proliferation, and degranulation of mast cells, and inhibits the proliferation of smooth muscle cells.

[0020] Figure 1 shows the results of the binding ELISA between the anti-mouse SCF antibody hybridoma culture medium and the mouse SCF antigen.

[0021] Figure 2 shows the SDS-PAGE Coomassie Blue staining results of the purified anti-mouse SCF antibody.

[0022] Figure 3 shows the isotyping ELISA results of the purified anti-mouse SCF antibody.

[0023] Figure 4 shows the results of the binding ELISA between the purified anti-mouse SCF antibody and the mouse SCF antigen.

[0024] Figure 5 shows the binding ELISA results for confirming cross-reactivity between the purified anti-mouse SCF antibody and the human SCF antigen.

[0025] Figure 6 shows the inhibitory activity of anti-mouse SCF antibody on Akt and ERK phosphorylation.

[0026] Figure 7 shows the results of the affinity verification of the anti-mouse SCF antibody against the mouse SCF antigen (SPR 1:1 binding model).

[0027] Figure 8 shows the results of a wound healing assay analyzing the cell proliferation inhibitory effect of anti-mouse SCF antibodies against mouse SCF in mouse endothelial cell MS-1 cell lines.

[0028] Figure 9 shows the results indicating that anti-SCF neutralizing antibodies completely block SCF-induced tube formation in hypoxic vascular endothelial cells. (A) Representative image of tubes formed in hypoxic mouse vascular endothelial cells and (B) results of quantitative analysis of tube formation. Tube formation in MS1 ​​cells was compared under hypoxic conditions with and without treatment with rm SCF (50 ng / mL) and anti-SCF neutralizing antibodies (1 or 10 mg / mL). Tube lengths were expressed as a relative change scale relative to the control group (Control; Cont). Data were expressed as mean ± SEM, and statistical analysis was performed using one-way ANOVA and Bonferroni post-hoc analysis (*p < 0.05, **p < 0.01, n = 4). Scale bar = 500 μm.

[0029] Figure 10 shows the results indicating that intratracheal administration of anti-SCF neutralizing antibodies inhibits the development of pulmonary arterial hypertension in mice. (A) Schematic diagram of the experimental protocol to evaluate the prophylactic effect of anti-SCF neutralizing antibodies in a mouse model of pulmonary arterial hypertension induced by chronic hypoxia and once-weekly SU5416 administration (hereinafter Hyp+SU). Mice reared under normal oxygen conditions were used as the control group. (B) Right ventricular systolic pressure (RVSP), mean right ventricular pressure (mRVP), right ventricular (RV) dP / dtmax, and heart rate measured via open-chest right heart catheterization, as well as cardiac output (CO) and right ventricular ejection fraction (RVEF) values ​​calculated via transthoracic echocardiography. Heart rates were similar across all groups (n = 5–11 mice per group). (C) WGA stained images of right ventricular tissue sections and the mean myocardial cell size measured in the corresponding sections (n ​​= 7–9). Scale bar = 20 μm. All data were expressed as mean ± SEM, and one-way ANOVA and Bonferroni post-hoc analysis were applied (*p < 0.05, **p < 0.01, ***p < 0.001).

[0030] The present invention aimed to discover therapeutic candidate substances that inhibit abnormal angiogenesis, inhibit differentiation, proliferation, and degranulation of mast cells, and inhibit proliferation of smooth muscle cells by producing an antibody that neutralizes SCF and inhibiting SCF / c-kit signaling. Accordingly, a monoclonal antibody that neutralizes SCF in mice was produced, and research results were obtained that neutralize SCF.

[0031]

[0032] The present invention provides a 2E7 antibody or an antigen-binding fragment thereof that neutralizes a mouse stem cell factor (SCF), comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 1, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 2, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 3; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 4, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 5, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 6.

[0033] Preferably, in the 2E7 antibody, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 7, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 8, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 2, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 9, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 3, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 10.

[0034] The above heavy chain variable region may include, but is not limited to, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 11, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 4, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 12, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 14.

[0035]

[0036] In addition, the present invention provides a 4B11 antibody or its antigen-binding fragment for neutralizing mouse SCF, comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 15, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 16, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 17; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 18, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 19, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 20.

[0037] Preferably, in the 4B11 antibody, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 21, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 15, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 22, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 16, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 23, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 17, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 24.

[0038] The above heavy chain variable region may include, but is not limited to, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 25, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 18, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 26, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 19, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 27, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 20, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 28.

[0039]

[0040] In addition, the present invention provides a 4E2 antibody for neutralizing SCF or an antigen-binding fragment thereof comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 29, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 30, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 31; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 32, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 33, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO. 34.

[0041] Preferably, in the 4E2 antibody, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 35, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 36, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 37, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 31, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 38.

[0042] The above heavy chain variable region may include, but is not limited to, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 39, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 32, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 40, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 33, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 41, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 34, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO. 42.

[0043]

[0044] In addition, the amino acid sequences of the entire light chain variable region and the entire heavy chain variable region of each of the above 2E7 antibody, 4B11 antibody, and 4E2 antibody were denoted by SEQ ID NOs 43 to 48, and the nucleotide sequences coding for each were denoted by SEQ ID NOs 49 to 54.

[0045]

[0046] In the present invention, the term “antibody” refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, comprising an immunoglobulin molecule that is immunologically reactive with a specific antigen, and may include, for example, monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. Additionally, the term “antibody” may include bivalent or bispecific molecules (e.g., bispecific antibodies), diabadies, triabadies, or tetraabadies.

[0047] In the present invention, the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same group of antibodies, and unlike polyclonal antibodies which can bind to multiple epitopes, such monoclonal antibodies exhibit single binding ability and affinity for a specific epitope. In the present invention, the term “full-length antibody” refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0048]

[0049] In the present invention, the term “heavy chain” may include both a full-length heavy chain and fragments thereof, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, a variable region VH, and three constant regions CH1, CH2, and CH3. Additionally, in the present invention, the term “light chain” may include both a full-length light chain and fragments thereof, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, a variable region VL, and a constant region CL.

[0050]

[0051] In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of the antibody of the present invention having the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0052]

[0053] The antibody of the present invention or its antigen-binding fragment may include not only the sequence of the antibody described herein but also biological equivalents thereof, to the extent that it can exhibit the ability to neutralize mouse SCF. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such changes include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. By analysis of the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Accordingly, based on this, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0054]

[0055] In addition, the present invention provides a nucleic acid molecule encoding the antibody or its antigen-binding fragment.

[0056] As used herein, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequence of a nucleic acid molecule encoding the heavy chain and light chain variable regions of the present invention may be modified, and said modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0057]

[0058] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0059] In the present invention, "vector" means a self-replicating DNA molecule used to carry a clonal gene (or another piece of clonal DNA).

[0060] In the present invention, “expression vector” refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism. The expression vector may preferably include one or more selectivity markers. The marker is a nucleic acid sequence having characteristics that can typically be selected by chemical methods, and includes any gene capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by a person skilled in the art.

[0061]

[0062] To express the DNA sequence of the present invention, any of the very diverse expression regulatory sequences may be used in the vector. Examples of useful expression regulatory sequences may include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTRs of retroviruses, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of fd code proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of said phosphatase, for example Pho5, promoters of the yeast alpha-mating system, and other sequences of configuration and induction known to regulate the expression of genes of prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof.

[0063] The vector expressing the antibody of the present invention may be either a vector system in which the light chain and heavy chain are simultaneously expressed in a single vector, or a system in which the light chain and heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells through co-transformation and targeted transformation. Co-transformation is a method of simultaneously introducing the respective vector DNA encoding the light chain and heavy chain into host cells and then selecting cells that express both the light chain and the heavy chain. Targeted transformation is a method of selecting cells transformed with a vector containing the light chain (or heavy chain), and then transforming the selected cells expressing the light chain back into a vector containing the heavy chain (or light chain) to finally select cells that express both the light chain and the heavy chain.

[0064]

[0065] In addition, the present invention provides a cell isolated by transforming it with a recombinant expression vector.

[0066] Cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the relevant art, and include, but are not limited to, prokaryotic host cells such as, for example, Escherichia coli, Bacillus subtilis and Bacillus churingensis strains, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0067] In the method for preparing the above-mentioned antibody or its antigen-binding fragment, the culture of the transformed cells may be carried out according to suitable media and culture conditions known in the relevant art. A person skilled in the art can easily adjust and use this culture process depending on the selected strain. Cell culture is classified into suspension culture and attachment culture according to the cell growth method, and into batch, fed-batch, and continuous culture methods according to the culture method. The media used for culture must adequately satisfy the requirements of the specific strain.

[0068]

[0069] In addition, the present invention provides a hybridoma cell line that produces the antibody or its antigen-binding fragment.

[0070] In the present invention, the monoclonal antibody may be produced from a hybridoma cell line. The hybridoma cells may be prepared using methods known in the art. For example, the hybridoma cells may be prepared by immunizing an animal with an antigen protein, fusing a splenocyte—an antibody-producing cell derived from the immunized animal—with a myeloma cell to produce a hybridoma, and then selecting a hybridoma that produces a monoclonal antibody that specifically binds to the antigen protein. The immunized animal may be an animal such as a goat, sheep, guinea pig, rat, or rabbit, in addition to the mouse used in the examples.

[0071]

[0072] In addition, the present invention provides a composition for detecting SCF antigens comprising the antibody or its antigen-binding fragment as an active ingredient.

[0073]

[0074] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of pulmonary arterial hypertension comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0075]

[0076] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier being one commonly used in formulations and including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition for preventing or treating cancer metastasis of the present invention may further comprise a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0077] The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, the oral composition may be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention may be administered by any device capable of transporting the active substance to target cells.

[0078] The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician who is normally skilled can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0079] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0080] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0081] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0082]

[0083] <Example 1> Antigen Immunity

[0084] An emulsion was prepared by mixing 50 μg of recombinant mouse SCF (Stem cell factor) protein (Cat. Cyt-275) purchased from Prospec with an equal volume of complete Freund's Adjuvant (Sigma, USA) (per mouse). The emulsion was injected into the peritoneal cavity of 7-week-old Balb / c mice. After 1 and 2 weeks, an emulsion mixed with incomplete Freund's Adjuvant (Sigma, USA) and the antigen was injected into the peritoneal cavity of the mice to perform a total of three immunizations and induce antibody production.

[0085]

[0086] <Example 2> Identification and Selection of Antibody-Generating Cells

[0087] Blood was collected from mice immunized using the above method, placed in a 1.5 mL microcentrifuge tube, and centrifuged at 13,000 rpm for 10 minutes. Antibody production was confirmed using the separated serum by performing an enzyme immunoassay. Three days prior to cell fusion, an emulsion mixed with incomplete Prawn Azuvant (Sigma, USA) and the antigen was injected once more into the peritoneal cavity of the mice.

[0088]

[0089] <Example 3> Preparation of Hybridoma

[0090] After confirming antibody production, mice were sacrificed to isolate splenocytes. The isolated B cells from the spleen were fused with mouse F / 0 myeloma cells. To perform cell fusion, the cultured F / 0 myeloma cells and the B cells isolated from the spleen of immunized mice were fused using a 50% polyethylene glycol 1450 (Sigma, Cat No. p7181, PEG, Poly Ethylene Glycol) solution. Subsequently, the hybridoma fused cells were suspended in 20% FBS DMEM medium in a pellet form after centrifugation. A feeder was pre-filled in a 96-well plate at a capacity of 100 μL / well, and the hybridoma suspension was dispensed at a rate of 100 μL / well. The plates were then cultured in a 37°C, 5% CO2 incubator.

[0091]

[0092] <Example 4> Selection and Isolation of Antibody-Generating Fusion Cells

[0093] To select antibody-producing fusion cells, hybridoma fusion cells were selected through HAT (final concentration of 100μM hypoxanthine, 0.4μM aminopterin, and 16μM thymidine).

[0094] The supernatant of the previously prepared fused cell culture medium was collected, and an ELISA (Enzyme Linked Immuno Sorbent Analysis) was performed to confirm whether antibodies specifically generated against the antigen used above were produced. The fused cell culture medium, which showed an absorbance at 450 nm of ELISA more than four times higher than the negative control group, was transferred to a 24-well plate for culture. Additionally, the culture medium was diluted to accommodate one cell per well in a 96-well plate, and the culture medium was recovered. An ELISA was then performed using a 96-well plate coated with recombinant human SCF or recombinant mouse SCF to select the fused cells that produced the final monoclonal antibody.

[0095]

[0096] <Example 5> ELISA on the culture medium of a monoclonal hybridoma producing mouse SCF antibodies

[0097] ELISA was performed to confirm the binding of anti-SCF antibodies produced from the hybridoma culture medium to the mouse SCF antigen and to verify whether the binding ability of each antibody was maintained at an equivalent level during the hybridoma culture process. Recombinant mouse SCF antigen protein was coated at a concentration of 20 ng / 100 μL / well and coated overnight in a 96-well plate at 4°C using a horizontal stirrer. Afterward, the coated solution was removed. With no remaining solution, 300 μL of 1X PBST containing 0.1% Tween 20 dissolved in 5% BSA was added per well, and the blocking reaction was carried out at 25°C for 1 hour. Subsequently, the solution was removed. For washing, the wells were placed in a squeeze bottle containing PBST with 0.1% Tween 20, and washing was performed three times to ensure a constant rate and volume. The antibody-producing hybridoma culture medium was prepared by mixing the undiluted culture medium with suspensions diluted with 1X PBS at concentrations of 1:10, 1:100, and 1:1000, placing 100 µL of each solution into each well of a 96-well plate. Mouse IgG 1 μg / mL and 1X PBS were used as the negative control and blank, respectively. The diluted suspensions of the culture mediums containing each prepared antibody were applied to two wells for duplication, and the reaction was carried out at 25°C for 2 hours. Afterward, the solution was drained. For washing, four washes were performed using PBST, ensuring a constant rate and volume. The 1-step Ultra TMB-ELISA substrate solution was equilibrated at RT for at least 30 minutes before use; 50 µL was applied to each well, and the reaction was carried out for 10 minutes. After 10 minutes, 50 µL of 1N H2SO4 solution was added to each well to stop the reaction. The results were obtained by measuring the absorbance at 450 nm using a SPECTROstar Nano Microplate reader.

[0098] As a result, it was confirmed in Figure 1 that all anti-mouse SCF antibodies maintained their binding ability to the mouse SCF antigen in the monoclonal hybridoma culture medium.

[0099]

[0100] <Example 6> SDS-PAGE and Coomassie Blue Staining Method for Size Confirmation of Purified Anti-Mouse SCF Antibody

[0101] To confirm the purity and size of the purified anti-mouse SCF antibody, SDS-PAGE was performed by running under 6% non-reducing SDS-PAGE and 10% reducing SDS-PAGE conditions. The results are shown in Figure 2.

[0102] Under reducing conditions, as shown in Figure 2, a band was observed showing a single clone with a heavy chain of 50 kDa and a light chain of 25 kDa or about 20 kDa.

[0103] For single clones 2C1, 2E7, 4B11, and 4E2, a heavy chain of 50 kDa was identified. For light chains, a light chain of 25 kDa was identified in single clones 2C1 and 4B11, and a light chain of less than 25 kDa was identified in single clones 2E7 and 4E2.

[0104] Specifically, there were 75 kDa 2B1, 2F10, 2E4, and 2D3 single clones possessing the heavy chain size of IgM. Similarly, even under non-reducing conditions, bands that failed to descend from the wells due to the large size of IgM were observed in the 2B1, 2F10, 2E4, and 2D3 single clones. In the remaining single clones, it was confirmed that they possessed bands with a purity of over 90% at 150 kDa.

[0105]

[0106] <Example 7> Isotyping ELISA of Purified Anti-Mouse SCF Antibody

[0107] An isotyping ELISA kit (Invitrogen, cat No. 37503) pre-coated with antigens capable of binding to mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, Kappa, and Lambda was used. For each row of the coated 96-well plate, 50 µL of purified anti-mouse SCF antibody, diluted to a concentration of 1 μg / mL in 1X PBS, was dispensed into each well. 50 µL of Goat Anti-Mouse IgG + IgA + IgM HRP Conjugate solution was then dispensed into each well. The reaction was carried out at 25°C for 1 hour. Afterward, the solution was drained. For washing, four washes were performed using PBST containing 0.1% Tween 20, ensuring a constant rate and volume. Finally, 75 µL of the TMB solution included in the kit was dispensed into each well, taking care to avoid introducing air bubbles. After a reaction of 5 minutes, the reaction was stopped by dispensing 75 µL of the stop solution included in the kit into each well, taking care to avoid introducing air bubbles. The results were obtained by measuring the absorbance at 450 nm using a SPECTROstar Nano Microplate reader.

[0108] As a result, among the monoclones 4E2, 4G12, 4B11, 4C11, 2B1, 2C1, and 2G10 for the purified anti-mouse SCF antibody in Figure 3, it was confirmed that monoclone 2B1 was IgM, monoclones 2C1 and 2G10 were IgG2a, and the remaining monoclones 4E2, 4G12, 4B11, and 4C11 were IgG1 heavy chains, and it was confirmed that the light chains were all kappa.

[0109]

[0110] <Example 8> Confirmation of SCF antigen binding ability of mouse SCF antibody

[0111] The binding affinity between the anti-mouse SCF antibody and the mouse SCF antigen was confirmed using ELISA.

[0112] ELISA was performed to confirm the binding of purified anti-mouse SCF antibodies to the mouse SCF antigen and to compare the binding abilities of each antibody. Recombinant mouse SCF antigen protein was coated at a concentration of 20 ng / 100 µL / well and coated overnight in a 96-well plate at 4°C using a horizontal stirrer. Afterward, the coated solution was removed. With no remaining solution, 300 µL of 1X PBST containing 0.1% Tween 20 dissolved in 5% BSA was added per well, and the blocking reaction was carried out at 25°C for 1 hour. Subsequently, the solution was removed. For washing, 1X PBST containing 0.1% Tween 20 was placed in a squeeze bottle, and washing was performed three times to ensure a constant rate and volume. Antibody suspensions were prepared by diluting the antibodies with 1X PBS to concentrations of 10, 5, 2, 1, 0.25, 0.0625, 0.015625, and 0.00390625 μg / mL, with 100 μL added to each well of a 96-well plate. Each prepared antibody dilution suspension was applied to two wells as a duplication, and the reaction was carried out at 25°C for 2 hours. Afterward, the solution was drained. For washing, PBST was applied at a constant rate and volume, and the plates were washed four times. The 1-step Ultra TMB-ELISA substrate solution was equilibrated at 25°C for at least 30 minutes before use, and 50 μL was added to each well to allow the reaction to proceed for 10 minutes. After 10 minutes, 50 μL of 1N H2SO4 solution was added to each well to stop the reaction. The results were obtained by measuring the absorbance at 450 nm using a SPECTROstar Nano Microplate reader.

[0113] In Figure 4, the binding affinity between the anti-mouse SCF antibody and the mouse SCF antigen is confirmed based on EC50 (Effective concentration 50%, μg / mL), with values ​​of 0.6045, 0.0749, 0.0958, 0.1564, 0.1034, 0.1041, 0.0251, and 0.0245 μg / mL for 4E2, 4G12, 4B11, 4C11, 2C1, 2G10, 2E7, and 2E10, respectively. Except for the 4E2 monoclonal, the values ​​for the others are similar, ranging from 0.0245 to 0.1564 μg / mL.

[0114]

[0115] The cross-reactivity between anti-mouse SCF antibodies and human SCF antigens was confirmed using ELISA.

[0116] ELISA was performed to confirm the binding of purified anti-mouse SCF antibodies to human SCF antigens and to compare the binding abilities of each antibody. Recombinant human SCF antigen protein was coated at a concentration of 20 ng / 100 μL / well and incubated overnight in 96-well plates at 4°C using a horizontal stirrer. Afterward, the coated solution was removed. With no remaining solution, 300 μL of 1X PBST containing 0.1% Tween 20 dissolved in 5% BSA was added per well, and the blocking reaction was carried out at 25°C for 1 hour. Subsequently, the solution was removed. For washing, PBST containing 0.1% Tween 20 was placed in a squeeze bottle, and washing was performed three times to ensure a constant rate and volume. Antibody suspensions were prepared by diluting the antibodies with 1X PBS to concentrations of 10, 5, 2, 1, 0.25, 0.0625, 0.015625, and 0.00390625 μg / mL, with 100 μL added to each well of a 96-well plate. Each prepared antibody dilution suspension was applied to two wells as a duplication, and the reaction was carried out at 25°C for 2 hours. Afterward, the solution was drained. For washing, four washes were performed using PBST, ensuring a constant rate and volume. The 1-step Ultra TMB-ELISA substrate solution was equilibrated at 25°C for at least 30 minutes before use, and 50 μL was added to each well to allow the reaction to proceed for 10 minutes. After 10 minutes, 50 μL of 1N H2SO4 solution was added to each well to stop the reaction. The results were obtained by measuring the absorbance at 450 nm using a SPECTROstar Nano Microplate reader.

[0117] In Figure 5, the cross-reactivity of anti-mouse SCF antibodies against human SCF antigens was confirmed based on EC50, with values ​​of 52.6932, 2.8488, 8.0533, 30.4273, 64.9363, 76.2879, 1.1595, and 1.2622 μg / mL for 4E2, 4G12, 4B11, 4C11, 2C1, 2G10, 2E7, and 2E10, respectively.

[0118]

[0119] <Example 9> Confirmation of Neutralizing Ability of Mouse SCF Antibody

[0120] The inhibitory effect of the anti-mouse SCF antibody on Erk and Akt phosphorylation induced by mouse SCF was confirmed through Western blot experiments on the mouse endothelial cell line MS-1.

[0121] SCF is known to induce phosphorylation of AKT and ERK 1 / 2. When mouse endothelial cells MS-1, known as a murine endothelial cell line, were treated with mouse SCF at a concentration of 200 ng / mL, an increase in phosphorylation of AKT and ERK 1 / 2 induced by SCF was confirmed.

[0122] Three x 10^5 cells were cultured in 60 mm cell culture dishes. One day later, the medium was changed to serum-free DMEM, a starvation medium, and the cells were cultured for 24 hours in a 37°C, 5% CO2 incubator to establish starvation conditions. After 24 hours, an amount of mouse SCF to achieve a final concentration of 200 ng / mL and an amount of anti-mouse SCF antibody to achieve a final concentration of 10 μg / mL were mixed into 100 μL of serum-free medium to a total volume and incubated at RT for 15 minutes. After 15 minutes, MS-1 cells were treated with 100 μL each of the SCF + antibody mixture, including a -SCF negative control and a +SCF positive control. The mixture was then incubated for 30 minutes at 37°C in a 5% CO2 incubator. It was placed in a cell culture medium and reacted.

[0123] Subsequently, cells were harvested using DPBS (Lonza, Switzerland) at 4°C. Cell lysis was immediately performed by adding cell lysis buffer, and a clear supernatant for total protein was obtained. Afterward, protein was quantified using a BCA assay to prepare an SDS-PAGE sample containing a reducing sample buffer, which was then boiled at 95°C for 7 minutes. A polyacrylamide gel containing 9% tris-glycine SDS was prepared, and the prepared reduced SDS-PAGE sample was dispensed into each well. SDS-PAGE was initiated by applying a voltage of 60V. After 30 minutes, the voltage was increased to 120V, and SDS-PAGE was terminated after approximately 1 hour and 50 minutes, until the tracking dye in the sample buffer was removed.

[0124] Transfer using a PVDF membrane was performed. The process was conducted in an ice box containing ice for 1 hour and 10 minutes under a voltage of 130V. The total protein transferred from the gel to the PVDF membrane was blocked with 5% BSA-TBST (1X TBS containing 0.2% Tween 20) for 1 hour at RT.

[0125] Subsequently, the primary antibodies were reacted overnight at 4°C using phospho-AKT (Cell Signaling Technology, 4060S, 1:1000), phosphor-ERK 1 / 2 (Cell Signaling Technology, 9101S, 1:2000), and GAPDH (Cell Signaling Technology, 2118S, 1:80,000) dissolved in 1% BSA-TBST. The secondary antibodies were diluted with anti-rabbit IgG-HRP conjugate (1:10000) in 1% BSA-TBST and reacted at RT for 1 hour.

[0126] After washing with TBST four times for 7 minutes as a washing process, the HRP reaction was carried out using Immobilion ECL Ultraplus Western HRP substrate (Millipore, Germany, WBULP-500mL) or Absignal detection kit (Abclon, Korea, ABC-3001).

[0127] While 4B11 was unable to inhibit the phosphorylation of Akt and ERK induced by SCF, antibodies 2E7 and 4E2 were shown to inhibit the phosphorylation of Akt and ERK induced by SCF (Fig. 6).

[0128]

[0129] <Example 10> Verification of the affinity of mouse SCF antibody to mouse SCF: SPR

[0130] Surface Plasmon Resonance (SPR) was performed to confirm the binding ability of anti-mouse SCF monoclonal antibodies 2E7, 2C1, 4B11, and 4E2 to mouse SCF. Using a Biacore 1K+ SPR system (Cytiva, USA), recombinant mouse SCF (Prospec, cyt-275) used for antibody production was immobilized onto a Sensor Chip CM5 (Cytiva, USA) at a level of 23.7 RU. Subsequently, each antibody was subjected to 2-fold serial dilution from concentrations ranging from 4000 nM or 2000 nM to 31.25 nM or 15.625 nM, and administered at each concentration including 0 nM. During this process, the flow rate of each antibody was set to 30 µL / min, the contact time to 240 seconds, and the dissociation time to 480 seconds. The analysis was performed using the Biacore Insight program K D The value was analyzed. K D The value is K d The value K a It is the value divided by the value; a lower value indicates a higher binding ability or affinity of the antibody for the antigen. The analysis model uses a 1:1 binding model and K D The value was calculated.

[0131] In Figure 7, for the 1:1 binding model, K D Based on the values, 2E7, 4B11, and 4E2 were found to be 3.51x10^(-8) M, 3.75x10^(-8) M, and 4.42x10^(-8) M, respectively.

[0132] Verification of the inhibitory ability of anti-mouse SCF antibody on mouse endothelial cell proliferation induced by mouse SCF in mouse endothelial cell line MS-1 (Wound healing assay)

[0133] Mouse endothelial cells MS-1 were seeded into a 6-well plate at a density of 5 x 10^5 cells and cultured in a 37°C, 5% CO2 incubator. After confirming the formation of a monolayer, a gap was created by drawing a straight line down the center of the well using a 200p yellow micropipette tip. Cell residue was removed through a washing process. Subsequently, the medium was changed to DMEM containing 0.5% FBS. Immediately, 100 µL of a mixture of 200 ng / mL SCF and 10 μg / mL anti-mouse SCF antibody, which had been incubated at 25°C for 15 minutes, was added. The cells were then cultured in a 37°C, 5% CO2 incubator, and after 48 hours, it was confirmed whether cell proliferation induced by mouse SCF was inhibited by the antibody.

[0134] In the negative control without SCF, minimal cell proliferation occurred due to 0.5% FBS, and gap closure of approximately 30% occurred after 48 hours, whereas it was confirmed that gap closure increased to 90% due to SCF. 4B11 showed no gap closure effect, while 2E7 and 4E2 showed gap closures of 45% and 54.25%, respectively, confirming that they significantly inhibited gap closure induced by SCF (Fig. 8).

[0135]

[0136] <Example 11> Inhibitory effect of 2E7 antibody on the development of pulmonary arterial hypertension

[0137] 1. Cell culture

[0138] Mouse vascular endothelial cells (MS1; ATCC, Manassas, VA, USA) were cultured in Endothelial Growth Medium-2 (Lonza, Walkersville, MD, USA) in a humidified 5% CO2 incubator at 37°C. Experiments under hypoxic conditions were performed in a modular incubator chamber (MIC-101; Billups-Rothenberg Inc., Del Mar, CA, USA) in an environment consisting of 94% N₂, 5% CO₂, and 1% O₂.

[0139]

[0140] 2. Tube forming ability test

[0141] MS1 cells were seeded into 24-well plates coated with Matrigel (Corning, Tewksbury, MA, USA). They were then cultured for 8 hours under hypoxic conditions in Endothelial Basal Medium (Lonza) alone containing 1% fetal bovine serum (Lonza), or in the same medium with the addition of recombinant mouse SCF (rm SCF; R&D Systems, Minneapolis, MN, USA) and / or anti-SCF neutralizing antibodies. The formed tube network was quantified by measuring the total length of the tubes.

[0142]

[0143] 3. Animals

[0144] All animal experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Additionally, all experimental procedures were approved by the Animal Ethics Committee of Chung-Ang University. Experimental animals were housed in individual ventilated cage racks equipped with micro-isolation cages, and the cages were lined with aspen sawdust (Northeastern Products Corp., Warrensburg, NY, USA). Animals were allowed free access to sterilized standard rodent food (LabDiet 5008, Purina, St. Louis, MO, USA) and maintained in an environment with a 12-hour light / 12-hour dark cycle. Anesthesia was induced during surgical procedures by intraperitoneal injection of ketamine (79.5 mg / kg) and xylazine (9.1 mg / kg), respectively.

[0145]

[0146] 4. Animal model of pulmonary arterial hypertension

[0147] Eight-week-old C57BL / 6 mice (Orient, Seoul, Korea) were exposed to a hypoxic environment of 10% O2 for three weeks, and 20 mg / kg of SU5416 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in dimethyl sulfoxide was injected intraperitoneally weekly. Control mice were maintained under normal oxygen conditions for the same period, but only dimethyl sulfoxide was injected intraperitoneally weekly. On the day hypoxia treatment began, phosphate-buffered saline (PBS, 100 μL) or anti-SCF neutralizing antibody (5 mg / kg, 100 μL) was injected into the trachea of ​​the mice using a microsprayer (BioJane, Shenzhen, China), and a second injection was administered 10 days later using the same method. Echocardiography and hemodynamic analysis were performed at three weeks after these treatments.

[0148]

[0149] 5. Cardiac ultrasound and hemodynamic measurements

[0150] Transthoracic echocardiography was performed using the VINNO 6 small animal ultrasound imaging system (VINNO Corporation, Suzhou, China). During the examination, mice capable of spontaneous breathing were lightly anesthetized by administering a minimal induction dose of isoflurane (Hana Pharmaceutical, South Korea). Echocardiographic images were recorded for subsequent quantitative analysis.

[0151] Hemodynamic parameters were measured via right heart catheterization in an open thoracotomy. Specifically, mice were anesthetized with 1.5% isoflurane and 100% O₂, intubated via a tracheotomy, and mechanical ventilation was performed. After opening the thoracotomy along the midline of the sternum, a 1.4-F micromanometer catheter (Millar Instruments, Houston, TX, USA) was inserted directly into the right ventricle. All data were collected and analyzed using the PowerLab data acquisition system (MPVS-Ultra Single Segment Foundation System, AD Instruments, Colorado Springs, CO, USA) and LabChart 8 (AD Instruments).

[0152]

[0153] 6. Quantification of myocardial cell size

[0154] Right ventricular tissue was fixed in paraffin, and sections were prepared. The sections were stained with WGA (Wheat Germ Agglutinin; Invitrogen, Carlsbad, CA, USA) conjugated with Alexa Fluor 488. The size of right ventricular cardiomyocytes was calculated by measuring the cross-sectional area of ​​the cardiomyocytes using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

[0155]

[0156] 7. Statistical Analysis

[0157] All data were expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). One-way ANOVA was conducted on all results, and Bonferroni post-hoc tests were applied. A p-value of less than 0.05 was considered statistically significant. The sample size (n) of the experiment used for each result is specified in the corresponding result.

[0158]

[0159] 8. Experimental Results

[0160] Anti-SCF neutralizing antibodies completely blocked SCF-induced tube formation in hypoxic endothelial cells.

[0161] Previous studies have revealed that SCF / cKIT signaling promotes tube formation in vascular endothelial cells under hypoxic conditions. To confirm the SCF-blocking effect of the anti-SCF neutralizing antibody, the present invention conducted a tube formation ability experiment on mouse vascular endothelial cells under hypoxic conditions. As a result, the anti-SCF neutralizing antibody completely blocked the increase in SCF-induced tube formation in mouse vascular endothelial cells under hypoxic conditions (Fig. 9).

[0162]

[0163] In addition, intratracheal administration of anti-SCF neutralizing antibodies in mice inhibited the development of pulmonary arterial hypertension.

[0164] To evaluate the therapeutic efficacy of anti-SCF neutralizing antibodies, a mouse model of pulmonary arterial hypertension induced by chronic hypoxia and once-weekly administration of SU5416 (hereinafter Hyp+SU) was constructed. During a 3-week exposure period to HyP+SU, mice were injected twice intratracheally with either PBS or anti-SCF neutralizing antibodies (Fig. 10A). Compared to normal control groups, right ventricle systolic pressure (RVSP), mean right ventricle pressure (mRVP), right ventricle (RV) dP / dtmax, and cardiac output (CO) increased significantly in Hyp+SU mice administered PBS, while right ventricle ejection fraction (RVEF) significantly decreased (Fig. 10B). In contrast, in Hyp+SU mice administered with anti-SCF neutralizing antibodies, RVSP, mRVP, RV dP / dtmax, and CO were significantly reduced, while RVEF was significantly increased to the level of normal control groups. To evaluate right ventricular hypertrophy (RVH) caused by pressure load on the right ventricle, the size of right ventricular cardiomyocytes was measured. The increase in cardiomyocyte size observed in Hyp+SU mice administered with PBS was significantly attenuated in the group administered with anti-SCF neutralizing antibodies (Fig. 10C). Taken together, these results demonstrate that intratracheal administration of anti-SCF neutralizing antibodies effectively prevents the development of pulmonary hypertension and right ventricular hypertrophy in a mouse model of pulmonary hypertension.

[0165]

[0166] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A 2E7 antibody or its antigen-binding fragment that neutralizes a mouse stem cell factor (SCF), comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 1, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 2, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 3; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 4, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 5, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO.

6.

2. In claim 1, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 7, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 1, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 8, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 2, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 9, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 3, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

10. A 2E7 antibody or its antigen-binding fragment for neutralizing mouse SCF, characterized in that the above-mentioned heavy chain variable region comprises a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 11, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 4, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 12, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 5, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 13, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

14.

3. A 4B11 antibody or its antigen-binding fragment for neutralizing mouse SCF comprising: a light chain variable region comprising a light chain CDR1 with an amino acid sequence represented by SEQ ID NO. 15, a light chain CDR2 with an amino acid sequence represented by SEQ ID NO. 16, and a light chain CDR3 with an amino acid sequence represented by SEQ ID NO. 17; and a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence represented by SEQ ID NO. 18, a heavy chain CDR2 with an amino acid sequence represented by SEQ ID NO. 19, and a heavy chain CDR3 with an amino acid sequence represented by SEQ ID NO.

20.

4. In paragraph 3, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 21, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 15, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 22, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 16, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 23, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 17, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

24. A 4B11 antibody or its antigen-binding fragment for neutralizing mouse SCF, characterized in that the above-mentioned heavy chain variable region comprises a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 25, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 18, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 26, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 19, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 27, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 20, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

28.

5. A 4E2 antibody or its antigen-binding fragment that neutralizes an SCF, comprising: a light chain variable region comprising a light chain CDR1 having an amino acid sequence represented by SEQ ID NO. 29, a light chain CDR2 having an amino acid sequence represented by SEQ ID NO. 30, and a light chain CDR3 having an amino acid sequence represented by SEQ ID NO. 31; and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence represented by SEQ ID NO. 32, a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO. 33, and a heavy chain CDR3 having an amino acid sequence represented by SEQ ID NO.

34.

6. In claim 5, the light chain variable region comprises a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 35, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 29, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 36, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 30, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 37, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 31, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

38. A 4E2 antibody or its antigen-binding fragment for neutralizing mouse SCF, characterized in that the above-mentioned heavy chain variable region comprises a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO. 39, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO. 32, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO. 40, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO. 33, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO. 41, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO. 34, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO.

42.

7. A nucleic acid molecule encoding the antibody of any one of paragraphs 1 to 6 or the antigen-binding fragment thereof.

8. A recombinant expression vector comprising the nucleic acid molecule of claim 7.

9. Cells isolated after being transformed with the recombinant expression vector of claim 8.

10. A hybridoma cell line that produces the antibody of any one of claims 1 to 6 or its antigen-binding fragment.

11. A composition for detecting SCF antigens comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6 as an active ingredient.

12. A pharmaceutical composition for the prevention or treatment of pulmonary arterial hypertension comprising, as an active ingredient, an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

Images