KR20160000881A - Composition for inhibiting myeloid-derived suppressor cells - Google Patents

Abstract

Disclosed herein is a novel use of a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the above peptide sequence, or a peptide thereof. Specifically, the use of said peptide for inhibiting myeloid-derived suppressor cells (MDSC) is disclosed. Inhibition of MDSC can solve the problem of suppression of immune response by MDSC. The peptides can be used in combination with other anticancer agents and adjuvants to effectively inhibit MDSC, thereby alleviating, treating or preventing diseases or conditions associated with MDSC. In addition, vaccines, particularly cancer vaccines, anti-cancer kits, or anticancer methods that do not have side effects associated with MDSC can be provided.

Description

Composition for inhibiting myeloid-derived suppressor cells (MDSC) <br> Description SureChem Title: Composition for inhibiting myeloid-derived suppressor cells

The present invention relates to novel compositions for inhibiting myeloid-derived suppressor cells (MDSC), MDSC inhibition kits, MDSC inhibition methods, anticancer compositions, anticancer kits, anticancer methods or telomerase peptides .

Myeloid-derived suppressor cells (MDSC) are a heterogeneous family of immature bone marrow cells stably differentiated by various tumor-secreting factors. MDSC inhibits the activity of cytotoxic T lymphocytes in a variety of ways.

The function of MDSC in vivo is in part to protect the organism from deleterious effects of excessive immune stimulation in acute and chronic infections and to limit the occurrence of autoimmune reactions to tissue antigens released by the trauma. Thus, it is beneficial for the action of MDSC to suppress the autoimmune response, but the proliferation of MDSC is also related to the pathological viewpoint. Activation is mediated through several transcription factors, resulting in the upregulation and expression of immunosuppressive factors such as ARG1 and NOS2 and increased production of NO, ROS, RNS and cytokines. In particular, the accumulation of MDSCs causes the immunosuppressed environment to persist so that the organism is constantly exposed to allergen and / or viral infections, which can lead to chronic inflammation. If the organism can not make an effective immune response, tissue damage may occur during chronic inflammation.

Only chemotherapeutic agents known to directly reduce MDSC in preclinical models are gemcitabine and 5-fluorouracil (5-FU). Gemcitabine has been reported to significantly reduce the number of MDSCs in the splenic gyrus in tumor-induced mice (Suzuki E., Kapoor V, Jassar AS, Kaiser LR, Albelda SM (2005) Gemcitabine selectively eliminates splenic Gr-1 + / CD11b + myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res 11: 6713-6721]. 5-FU is also known to significantly reduce the percentage of MDSC, which is said to be greater than gemcitabine.

Another way to inhibit MDSC is to add miR-142 and / or miR-223 ribonucleotides to MDSC (WO 2013/082591). miR-142 and / or miR-223 ribonucleotides can reduce the number of MDSCs by allowing them to differentiate into macrophages, dendritic cells, and the like.

As another method for inhibiting MDSC, a method of using a bisphosphonate or CCR2 inhibitor as an adjuvant is known (WO 2011/116299). Bisphosphonates include, but are not limited to, claudonate, zoledronate, palmyrronate, etidronate, or other bisphosphonate drugs. CCR2 inhibitors include RS 1028595 or PF-04178903.

On the other hand, a granulocyte-macrophage colony-stimulating factor (GM-CSF), which is adjuvanted with the vaccine in preclinical studies, is known to increase MDSC in tumor micro-environments [Curran MA, Allison JP (2009) Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69: 7747-7755]. In addition, it has been known that the use of GM-CSF as a vaccine adjuvant in a clinical trial increases the number of MDSCs in the blood (Filipazzi P, Valenti R, Huber V, Pilla L, Canese P, Iero MC, Mariani L, Parmiani G , Rivoltini L et al. (2007) Identification of a new subset of myeloid suppressor cells in peripheral blood melanoma patients with a granulocyte-macrophage colony-stimulating factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553].

CD11c, F4 / 80, and MHCII are known to be indicators of the maturation of MDSC. CD11c is a transmembrane protein expressed on the surface of most dendritic cells. F4 / 80 is a homologue protein of EMR1 (Egf-like module containing, mucin-like hormone receptor-like1) and is a transmembrane protein expressed on the cell surface of human macrophages. MHC II (MHC class II) is an extracellular protein normally expressed only in antigen-presenting cells such as dendritic cells.

[Prior Art Literature]

[Patent Literature]

(1) WO 2013/082591

(2) WO 2011/116299

[Non-Patent Document]

(1) Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM (2005) Gemcitabine selectively eliminates splenic Gr-1 + / CD11b + myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res 11: 6713-6721

(2) Curran MA, Allison JP (2009) Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69: 7747-7755

(3) Filipazzi P, Valenti R, Huber V, Pilla L, Canese P, Iero MC, Mariani L, Parmiani G, and Rivoltini L et al (2007) Identification of a new subset of myeloid suppressor cells in peripheral blood melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553

In one aspect, an object of the present invention is to inhibit MDSC.

In another aspect, the object of the present invention is to activate immunity.

In another aspect, the object of the present invention is to activate immunity which is inhibited by MDSC.

In another aspect, the object of the present invention is to solve the problem that the immune response of the vaccine is inhibited by MDSC.

In another aspect, the object of the present invention is to solve the problem that the immune response of cancer vaccine is inhibited by MDSC.

In another aspect, the object of the present invention is to alleviate, treat or prevent diseases or conditions associated with MDSC.

In another aspect, an object of the present invention is to provide a chemotherapeutic composition, an anti-cancer kit, or an anticancer method which has no side effects of lowering the immune response according to MDSC.

In another aspect, an object of the present invention is to provide a cancer vaccine which has no side effects of lowering the immune response according to MDSC.

In one aspect, the present invention provides a composition for inhibiting bone marrow-derived inhibitory cells (MDSC) comprising a peptide comprising SEQ ID NO: 1 as an active ingredient for inhibiting MDSC, a peptide having 80% or more sequence homology with the peptide sequence Peptide, or peptide fragment thereof, wherein the peptide is contained in an amount effective to inhibit MDSC, wherein the peptide is a composition for inhibiting bone marrow-derived inhibitory cells.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In one aspect, the invention may be a kit for inhibiting MDSC. The kit comprises a peptide comprising SEQ ID NO: 1 as an active ingredient for inhibiting MDSC, a peptide having a sequence homology of at least 80% with the peptide sequence, or a fragment thereof, wherein the peptide is effective against MDSC inhibition An amount of a composition for inhibiting MDSC; Anticancer agents; And instructions. In one aspect, the kit may further comprise an adjuvant.

In one aspect, the MDSC inhibitor kit may be to ameliorate, prevent, or treat a disease or condition caused by MDSC through MDSC inhibition.

In another aspect, the MDSC inhibition kit may be to ameliorate, prevent, or treat cancer through MDSC inhibition.

In one aspect, the present invention provides a composition comprising a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a peptide thereof; Anticancer agents; And an anti-cancer kit comprising the instructions. The kit may further comprise an adjuvant composition.

In one aspect, the invention provides a method of inhibiting bone marrow-derived suppressor cells comprising administering to a subject in need thereof a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with said peptide sequence, Or a fragment thereof, to a subject in need of MDSC inhibition. In one aspect, the method may further comprise administering an anti-cancer agent in combination with the peptide. In another aspect, the method may further comprise administering an adjuvant in combination with the peptide.

In another aspect, the present invention provides a method of improving, treating or preventing cancer comprising administering an effective amount of a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, Or prophylactic treatment, comprising administering to a subject in need of improvement, treatment or prevention of cancer in combination with an anticancer agent and / or adjuvant.

In one aspect, the present invention provides a composition for inhibiting bone marrow-derived inhibitory cells, comprising a peptide comprising SEQ ID NO: 1, a peptide having 80% or more of sequence homology with the peptide sequence, or a fragment thereof Lt; / RTI &gt;

In one aspect, the present invention can inhibit MDSC.

In another aspect, the invention can activate immunity.

In another aspect, the present invention can activate immunity that is inhibited by MDSC.

In another aspect, the present invention can solve the problem that the immune response of the vaccine is inhibited by MDSC.

In another aspect, the present invention can solve the problem that the immune response of cancer vaccine is inhibited by MDSC.

In another aspect, the invention can alleviate, treat, or prevent a disease or condition associated with MDSC.

In another aspect, the present invention can provide an anti-cancer composition, an anti-cancer kit, or an anti-cancer method without side effects of lowering the immune response according to MDSC.

In another aspect, the present invention can provide a cancer vaccine that does not have side effects of lowering the immune response according to MDSC.

FIG. 1 is a graph showing the expression of CD11c in PEP1 and the comparative substance-treated groups in PMN-MDSC (polymorphonuclear pulmonary fibroblast cell) type as raw data of Mean Fluorescence Intensity (MFI).
2 is a graph showing the expression of F4 / 80 marker in the PEP1 and the comparative substance-treated groups in the PMN-MDSC type as the absolute value of the mean fluorescence value.
Figure 3 is a graph showing the expression of MHC II markers in absolute values of mean fluorescence values in PEPl and the comparative substance treated groups in the Mo-MDSC (mononuclear leukemia cell line) type.
4 is a graph showing CD11c expression in the PMN-MDSC type in PEP1 and the comparative substance-treated group as a relative value of the mean fluorescence value.
FIG. 5 is a graph showing the expression of F4 / 80 marker in the PEP1 and the comparative substance-treated groups in the PMN-MDSC type as the relative value of the mean fluorescence value.
FIG. 6 is a graph showing the expression of MHC II marker in the PEP1 and the comparative substance-treated groups in the PEP1 type in the Mo-MDSC type as a relative value of the mean fluorescence value.
Figure 7 is a graph showing the relationship between the levels of MDSC and proinflammatory cytokines.
FIG. 8 is a graph showing changes in MDSC of patient group (Comparative Example 2, arm 2) receiving only gemcitabine plus capecitabine and GemCap and PEP1 (Example 3, arm 3) FIG.

All publications mentioned herein are incorporated by reference in their entirety.

As used herein, the term " MDSC inhibition "is a concept including not only reducing the number of MDSC but also inhibiting the activity of MDSC. Reducing the number not only inhibits the production of cells but also includes killing already produced cells or differentiating them into other cells. In addition, all mechanisms referred to as "inhibition" in biological terms are included.

As used herein, "combination administration" or "combined administration" refers not only to simultaneous administration in terms of physical time, but also in combination with two or more drugs depending on each drug-specific administration schedule to be combined &Lt; / RTI &gt;

The present inventors have found that MDSC is significantly increased in cancer patients. It has also been found that MDSC can inhibit the effect of a cancer vaccine administered for the treatment of cancer patients, which may render cancer treatment impossible. Therefore, it is necessary to reduce the increased MDSC in cancer patients for cancer treatment through cancer vaccine.

We have also found that when a combination of two chemotherapeutic agents, gemcitabine, and 5-fluorouracil, 5-FU, or capecitabine, which are known to directly reduce MDSC, , But did not decrease MDSC unlike the single use. Rather, a significant number of patients were found to have elevated MDSC. This can be a major limitation for the combined treatment of gemcitabine and 5-fluorouracil (or capecitabine).

The use of the peptides disclosed herein can significantly reduce MDSC. By reducing MDSC, a set of pathological problems associated with MDSC can be solved.

The peptides disclosed herein may be some fragments of telomerase or analogs thereof. Telomere is a genetic material that is repeatedly present at the end of a chromosome, and is known to prevent damage to the chromosome or its binding to other chromosomes. As the cell divides, the length of the telomere is getting shorter. When there is more than a certain number of cell divisions, the telomere becomes very short, and the cell stops dividing and dies. On the other hand, it is known that lengthening the telomeres prolongs the life of the cells. For example, it is known that the cancer cells secrete an enzyme called telomerase and prevent the shortening of the telomeres.

The peptide disclosed herein may comprise a peptide having a sequence of SEQ ID NO: 1 or a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a fragment thereof having a sequence of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% , 98% or more, 99% or more of sequence homology.

In one aspect of the invention, the peptide comprises a telomerase, specifically a peptide derived from human ( Homo sapiens ) telomerase.

The peptides disclosed herein may comprise a peptide comprising SEQ ID NO: 1 or a peptide having a sequence homology with the fragment. Also, the peptide disclosed in the present specification can be prepared by reacting a peptide or a fragment thereof comprising SEQ ID NO: 1 with one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, Or 7 or more amino acid altered peptides.

In one aspect of the invention, the peptide can be composed of up to 30 amino acids.

The peptides shown in SEQ ID NO: 1 are shown in Table 1 below. The "name" in Table 1 below is what the peptides are named for. In one aspect of the invention, the peptide of SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having a sequence of SEQ ID NO: 1, a peptide of a sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the above peptide sequence, And "synthetic peptide" synthesized by selecting peptides at positions. SEQ ID NO: 2 shows the amino acid sequence of the whole telomerase.

In one aspect of the invention, the amino acid change is of a property that causes the physicochemical properties of the peptide to change. For example, amino acid changes such as improving the thermal stability of the peptide, altering the substrate specificity, changing the optimum pH, etc. can be performed.

As used herein, the term "amino acid" includes D-isomers and modified amino acids as well as the 22 standard amino acids that are naturally incorporated into the peptide. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. In another aspect of the present invention, the peptide may include post-translationally modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (such as, for example, beta-depleted deamidation , Deamidation) and structural changes (e.g., formation of a disulfide bridge). Also included are amino acid changes such as changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that take place during binding with crosslinkers to form peptide conjugates.

The peptides disclosed herein may be wild type peptides identified and isolated from natural sources. Alternatively, the peptide disclosed herein may be an artificial variant comprising an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted as compared to a peptide of SEQ ID NO: 1 or a fragment thereof. Amino acid changes in wild-type polypeptides as well as in artificial variants include conservative amino acid substitutions that do not significantly affect folding and / or activity of the protein. Examples of conservative substitutions include, but are not limited to, basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine) Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring interactions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

In one aspect, the present invention provides a composition for inhibiting myeloid-derived suppressor cells (MDSC), wherein the composition comprises a peptide comprising SEQ ID NO: 1 as an active ingredient for inhibiting MDSC, a peptide comprising 80% Or a fragment thereof, and the peptide is a composition for inhibiting myeloid-derived suppressor cells (MDSC), which is contained in an amount effective to inhibit MDSC.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In another aspect, the present invention provides a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And an adjuvant.

In one aspect, the invention may be a kit for inhibiting MDSC. The kit comprises the aforementioned composition for inhibiting MDSC; Anticancer agents; And instructions. In one aspect, the kit may further comprise an adjuvant.

In one aspect, the MDSC inhibitor kit may be to ameliorate, prevent, or treat a disease or condition caused by MDSC through MDSC inhibition.

In another aspect, the MDSC inhibition kit may be to ameliorate, prevent, or treat cancer through MDSC inhibition.

In one aspect, the present invention provides a composition comprising a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a peptide thereof; Anticancer agents; And an anti-cancer kit comprising the instructions. The kit may further comprise an adjuvant composition.

In the above-mentioned kits, the description may include a description of how the MDSC inhibiting composition comprising the peptide is used in combination with an anticancer agent. Specifically, it may include a description of the dose, administration time, administration method, etc. of each drug. In addition, the instructions may include uses for inhibiting MDSC, anticancer or cancer vaccine use. Specifically, any disease or condition that may be required to inhibit MDSC may be included. In addition, the instructions may contain side effects and cautions for administration.

In one aspect, the invention provides a method of inhibiting bone marrow-derived inhibitory cells (MDSC) comprising administering to a subject in need thereof an effective amount of a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, Or a peptide fragment thereof, to a subject in need of MDSC inhibition. In one aspect, the method may further comprise administering an anti-cancer agent in combination with the peptide. In another aspect, the method may further comprise administering an adjuvant in combination with the peptide.

In another aspect, the present invention provides a method of improving, treating or preventing cancer comprising administering an effective amount of a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, Or prophylactic treatment, comprising administering to a subject in need of improvement, treatment or prevention of cancer in combination with an anticancer agent and / or adjuvant.

In one aspect, the cancer can be, but is not limited to, renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, lung cancer, blood cancer, prostate cancer, adenocarcinoma, prostate cancer or pancreatic cancer.

In one aspect, the present invention provides a composition for inhibiting bone marrow-derived inhibitory cells, comprising a peptide comprising SEQ ID NO: 1, a peptide having 80% or more of sequence homology with the peptide sequence, or a fragment thereof Lt; / RTI &gt;

In the present specification, the bone marrow-derived inhibitory cell (MDSC) may be a bone marrow-derived inhibitory cell of an individual having a tumor. In addition, the composition may be for administration to an individual in need of inhibiting MDSC. MDSC inhibits the activity of cytotoxic T lymphocytes (T lymphocytes) and thus functions to suppress immunity. Although there is a net function of suppressing unnecessary excessive immune responses such as autoimmunity, there is also the reverse function of suppressing immunity in the situation where an immune response is needed to cause disease, exacerbate or prevent proper treatment. For example, MDSC has been increased in tumor or cancer patients, which disables the efficacy of the cancer vaccine by significantly reducing the effect of the cancer vaccine administration. In this situation, effectively reducing the number of MDSCs will enable cancer treatment to be carried out smoothly and effectively.

Diseases or conditions caused by MDSC herein are apparent to those skilled in the art. Diseases or conditions caused by MDSCs referred to herein include both diseases or conditions caused by MDSCs known in the art. Bone diseases such as osteolytic bone disease; Multiple myeloma; Glioblastoma; Infections: bacterial & parasitic infections such as bacterial or parasitic infections; Acute or chronic inflammation; Traumatic stress; Sepsis and transplantation; Autoimmunity such as intraocular autoimmune diseases; Inflammatory bowel disease, Cancer cachexia; Or tuberculosis (TB).

MDSCs herein may include all MDSCs regardless of their phenotype. The various phenotypes of MDSC are known in the art. For example, the phenotype is selected from CD15, IL4Ra, CD14, CD11b, HLA-DR, CD33, Lin, FSC, DR and SSC and optionally CD45, CD18, CD80, CD83, CD86, HLA-I and survival / . For example, the phenotype is MDSC1 based on the label IL4Ra + and CD14 +; MDSC2 based on the label IL4Ra + and CD15 +; MDSC1 and MDSC2 based on the label IL4Ra +; MDSC3 based on the markers Lin-, HLA-DR- and CD33 +, and optionally CD18 + and HLA-I +; MDSC4 based on the label CD14 +, HLA-DR (- / lo), FSChi and SSCim; MDSC5 based on the labeled CD11b +, CD14- and CD15 +, and optionally FSChi, SSCim, CD80-, CD83-, CD86- and HLA-DR-; MDSC6 based on CD15 +, SClo and SSChi; And MDSC based on Lin, DR and CD11b +. For example, the phenotype may be Lin-DR-CD11b +.

The concentration of the peptides in the compositions disclosed herein can be routinely determined as is known in the art. For example, a composition according to one aspect of the present invention comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a peptide thereof, / L, specifically from 0.1 g / L to 100 g / L, more specifically from 1 g / L to 10 g / L, but it can be suitably controlled when the effect varies depending on the dose. When it is contained in the above-mentioned range or below, it is not only suitable for exhibiting the intended effect of the present invention but also can satisfy both the stability and safety of the composition and may be suitably included in the above range in terms of cost effectiveness .

The dose, the method of administration, the period of administration, and the like of the peptide are well known in the art and can be administered according to criteria known in the art depending on the condition of each patient. Specific dosage determinations are within the level of those skilled in the art, and their daily dose is, for example, from 0.1 ng / kg / day to 10 mg / kg / day, more specifically from 0.1 to 1 mg / kg / day, more specifically from 1 ug / kg / day to 100 ug / kg / day, and more specifically from 2 ug / kg / day to 50 ug / kg / day, The age of the subject, the health condition, complications, and the like. For example, the peptide may be administered via intradermal administration. The dosing interval may be administered once a day at intervals of two days, and the interval between dates may be extended over time. The first week is administered three times a week (days 1, 3 and 5), and once every 2, 3, 4, and 6 weeks (every 8, 15, 22, and 36 days) . The single dose may be from 0.1 to 3 mg on an adult basis. The dose may be at least 0.1 mg, at least 0.2 mg, at least 0.3 mg, at least 0.4 mg, at least 0.45 mg, or at least 0.5 mg. The dose may be 3 mg or less, 2.5 mg or less, 2.0 mg or less, 1.5 mg or less, 1.0 mg or less, 0.9 mg or less, 0.8 mg or less, 0.7 mg or less, or 0.6 mg or less.

In one aspect, the peptides described herein may be administered in combination with an anti-cancer agent. Anticancer agents include, but are not limited to, chemotherapeutic agents and biotherapeutic agents. In particular, it can be a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, DNA alkylating agents, anti-metabolites, natural products, hormones, and the like. The chemotherapeutic agent may be a metabolic antagonist. Metabolic antagonists refer to a group of molecules that interfere with DNA and RNA synthesis. Most metabolic antagonists have similar structures to building blocks of DNA and RNA. Subtypes of metabolic antagonists include, but are not limited to, anti-folates, fluoropyrimidines, deoxynucleoside analogues, and thiopurines. Fluoropyrimidines include, but are not limited to, fluorouracil and capecitabine. Capecitabine is a prodrug of 5-fluorouracil. The deoxynucleoside analogues may be selected from the group consisting of cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine, But are not limited to, cladribine, clofarabine, and pentostatin. A chemotherapeutic agent may be one in which two or more agents are administered in combination. For example, a fluoropyrimidine and a deoxynucleoside analog may be administered in combination. In another embodiment, gemcitabine and 5-fluorouracil (or its prodrug, capecitabine) may be administered in combination. In another embodiment, gemcitabine and capecitabine may be administered in combination.

Appropriate doses of the anticancer agents mentioned herein are already well known in the art and can be administered according to criteria known in the art, depending on the condition of each patient. The specific dosage determination is within the level of ordinary skill in the art, and its daily dose is, for example, from 1 μg / kg / day to 10 g / kg / day, more specifically from 10 μg / kg / day to 10 mg / kg / day, more specifically 50 ug / kg / day to 10 mg / kg / day, depending on various factors such as the age, .

For example, in the case of gemcitabine, it may be administered intravenously every week, the dose may be in the range of 100 to 10,000 mg / m ² once a week, the dosing cycle may be every 2 to 8 weeks, The number of times of administration can be 1 to 6 times, and can be released for the last 1 to 4 weeks of each administration cycle. For example, intravenous doses may be administered weekly for the first 3 weeks during the 4-week dosing cycle and may be withdrawn for the remaining 1 week. Specifically, per dose is 100 mg / m ² or more, 200 mg / m ² or more, 300 mg / m ² or more, 400 mg / m ² or more, 500 mg / m ² or more, 600 mg / m ² or more, 700 mg / m ^2, greater than 800 mg / m ^2, or greater than 900 mg / m ² . Alternatively, administration of 10,000 mg / m ² or less amount, 9000 mg / m ² or less, 8000 mg / m ² or less, 7000 mg / m ² or less, 6000 mg / m ² or less, 5000 mg / m ² or less, 4000 mg / m ² or less, 3000 mg / m ² or less, 2000 mg / m ² or less, 1500 mg / m ² or less, 1400 mg / m ² or less, 1300 mg / m ² or less, 1200 mg / m ² or less, 1100 mg / m ² hereinafter, 1050 mg / m ² or less, 1030 mg / m ² or less, 1020 mg / m ² or less, or may be up to 1010 mg / m ^2. The dosing cycle may be 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. The cycle of administration may be at least once, at least two times, at least three times, at least four times, or at least five times. The duration of administration during each administration cycle may be greater than 1 week, greater than 2 weeks, greater than 3 weeks, greater than 4 weeks, greater than 5 weeks, or greater than 6 weeks. The period of abstinence during each dosing cycle may be more than the last 1 week, the last 2 weeks, the last 3 weeks, or the last 4 weeks or more.

In the case of capecitabine, it can be administered orally. The administration interval may be twice a day. The dosage is 500 mg / m ² or more, 600 mg / m ² or more, 700 mg / m ² or more, 800 mg / m ² or more, 900 mg / m ² , 1,000 mg / m ² , ² , 1,200 mg / m ² , 1,300 mg / m ² , 1,400 mg / m ² , 1,500 mg / m ² , 1,600 mg / m ^{2 or} more. Alternatively, the dosage amount of 15,000 mg / m ² or less, 12,000 mg / m ² or less, 10,000 mg / m ² or less, 9000 mg / m ² or less, 8000 mg / m ² or less, 7000 mg / m ² or less, 6000 mg / m ² or less, 5000 mg / m ² or less, 4000 mg / m ² or less, 3000 mg / m ² or less, 2000 mg / m ² or less, 1,900 mg / m ² or less, 1,800 mg / m ² or less, 1,700 mg / m ² &Lt; / RTI &gt; The daily dose may be administered in two or more divided doses.

In one aspect, the peptides and / or anti-cancer agents described herein may be administered in combination with an adjuvant. The adjuvant may be an adjuvant that increases MDSC. From an immunological point of view, the adjuvant is added to the vaccine to stimulate an immune response to the target antigen, but does not itself provide immunogenicity. All vaccines induce inflammation, resulting in the accumulation of bone marrow cells (monocytes and neurotrophs). Some of these cells are MDSC. There are also adjuvants that are added to stabilize formulations of the vaccine for purposes other than immune response stimulation. Immunological adjuvants are well known in the art [J Biomed Biotechnol. 2012; 2012: 831486. Published online Mar 13, 2012]. Immunological adjuvants include inorganic adjuvants such as aluminum salts and organic adjuvants such as oil based, virosome, squalane. Organic adjuvants include, but are not limited to, emulsions, microbial-derived, synthetic adjuvants, cytokines, and the like. Nine species of cytokine adjuvants are known. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF), which activates mature granulocytes and macrophages, is mainly used for hepatitis B, HIV and cancer vaccines [J Biomed Biotechnol. 2012; 2012: 831486. Published online Mar 13, 2012]. The adjuvant herein may be an adjuvant that increases MDSC. The use of adjuvants that increase MDSC will inhibit the immune response and thus prevent the vaccine from being effective. In such a situation, a means to prevent the increase of the MDSC of the adjuvant used is needed.

Appropriate doses of the adjuvants mentioned herein are well known in the art and can be administered according to criteria known in the art according to the condition of each patient. The specific dosage determination is within the level of ordinary skill in the art, and its daily dose is, for example, from 1 μg / kg / day to 10 g / kg / day, more specifically from 10 μg / kg / day to 10 mg / kg / day, more specifically 50 ug / kg / day to 10 mg / kg / day, depending on various factors such as the age, .

For example, in the case of GM-CSF, a dose of 7 to 700 mg on an adult basis is administered before the administration of the peptide disclosed herein, for example, 1 minute to 150 minutes before, 5 to 80 minutes before, or 10 to 15 minutes before &Lt; / RTI &gt; The administration time may be 1 minute or more before, 3 minutes or more before, 5 minutes or more before, 7 minutes or more before, 8 minutes or more before, 9 minutes or 10 minutes or more before administration of the peptide. Before 150 minutes, before 130 minutes, before 110 minutes, before 100 minutes, before 90 minutes, before 80 minutes, before 70 minutes, before 60 minutes, before 50 minutes, after 40 minutes It can be administered before, 30 minutes, less than 20 minutes, or less than 15 minutes before. The dose may be 7 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 60 mg or more, or 70 mg or more. The dose may be less than 700 mg, less than 600 mg, less than 500 mg, less than 400 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg or less than 80 mg.

GM-CSF, an adjuvant added to vaccines in preclinical studies, has been shown to increase MDSC in tumor microenvironment [Curran MA, Allison JP (2009) Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69: 7747-7755]. In addition, it has been known in clinical practice that the use of GM-CSF as a vaccine adjuvant at low doses increases the number of MDSCs in the blood [Filipazzi P, Valenti R, Huber V, Pilla L, Canese P, Iero MC, Mariani L, G, Rivoltini L et al. (2007) Identification of a new subset of myeloid suppressor cells in peripheral blood melanoma patients with modulation of a granulocyte-macrophage colony-stimulating factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553]. The adjuvant added to aid the immune function of the vaccine, rather than increasing the number of MDSCs, interferes with the immune response. Thus, GM-CSF has a large limitation that it is difficult to use as adjuvant for cancer vaccine.

The inventors have uncovered an epoch-making method to solve the problem of loss of MDSC reduction ability by combination of gemcitabine with 5-fluorouracil (or capecitabine) and the problem of increase of MDSC in tumor micro-environment of GM-CSF I got it. We have found that Lin-DR-CD11b + MDSC is not increased in patients receiving GemCap and PEPl at the same time when administering a low dose of GM-CSF as the adjuvant of PEP1.

The high% MDSC before vaccination does not make it possible to cause an immune response against tumor-associated antigens.

In one aspect of the invention, the composition may be a pharmaceutical, cosmetic or food composition.

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

The pharmaceutical composition according to one aspect of the present invention can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramuscularly, intradermally or subcutaneously.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Formulations for parenteral administration may be, but are not limited to, injections, drops, lozenges, ointments, gels, creams, suspensions, emulsions, suppositories, patches or spraying agents.

The pharmaceutical composition according to one aspect of the present invention may contain additives such as a diluent, an excipient, a lubricant, a binder, a disintegrant, a buffer, a dispersant, a surfactant, a colorant, a fragrance or a sweetener as necessary. The pharmaceutical composition according to one aspect of the present invention can be prepared by a conventional method in the art.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The abbreviated term in front of a noun is not intended to limit the quantity but to indicate that there is more than one noun item mentioned. The terms " comprising ", "having ", and" containing "are to be construed as open (i.e., meaning including but not limited to).

To refer to a range of values is an easy way to substitute for referring individually to each distinct value falling within that range and each separate value that is not explicitly stated is referred to individually in the specification Are incorporated herein by reference. All range end values are contained within that range and can be combined independently.

All methods mentioned herein may be performed in any suitable order unless otherwise indicated or clearly contradicted by context. It is to be understood that the use of any embodiment and all of the embodiments or example language (e.g., "such as ") is for the purpose of describing the present invention only, It is not intended to be limiting. No language in the specification should be construed as obliging any non-claimed components to practice the present invention. Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferred embodiments of the present invention include the most optimal mode known to the inventors for carrying out the present invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately utilize such variations, and the inventors expect the invention to be practiced otherwise than as described herein. Accordingly, the present invention includes equivalents and all modifications of the subject matter of the invention as recited in the appended claims, as permitted by the patent law. Moreover, any combination of the above-mentioned components within all possible variations is included in the present invention unless otherwise specified or contradicted by context. While the present invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will readily appreciate that many changes can be made in form and detail without departing from the spirit and scope of the invention as defined by the following claims .

DETAILED DESCRIPTION OF THE INVENTION

Example 1 Synthesis of Peptide (PEP1)

A peptide (PEP1) comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof is prepared according to a conventional solid phase peptide synthesis method. Specifically, the peptides were synthesized by coupling one amino acid from the C-terminal through Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). The first amino acid at the C-terminus of the peptides attached to the resin was used as follows. For example:

NH ₂ -Lys (Boc) -2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg (Pbf) -2-chloro-Trityl Resin

All amino acid sources used for peptide synthesis are Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl). For example:

Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg- Fmoc-Lys (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Met-OH, Fmoc- -Asn (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

As the coupling reagent, HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] Respectively. Fmoc removal was performed using piperidine in DMF in 20% DMF. Cleavage Cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5] was used to remove the synthetic peptides from the resin and to remove the protecting groups of the residues. Respectively.

Each of the peptides was synthesized by repeating the steps of reacting corresponding amino acids with a starting amino acid having an amino acid protecting group bonded thereto on a solid support, washing with a solvent, followed by deprotection. The synthesized peptide was cleaved from the resin and purified by HPLC. The synthesis was confirmed by MS and lyophilized.

A detailed procedure is as follows, taking Pep 1 (EARPALLTSRLRFIPK) composed of SEQ ID NO: 1 as an example.

1) Coupling

The protected amino acid (8 eq) and the coupling reagent HBTU (8 eq) / HOBt (8 eq) / NMM (16 eq) were dissolved in DMF and added to NH ₂ -Lys (Boc) -2-chloro-Trityl Resin , And reacted at room temperature for 2 hours and washed with DMF, MeOH and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF in 20% DMF was added, and the reaction was carried out at room temperature for 5 minutes twice, followed by washing with DMF, MeOH and DMF.

3) By the reaction of 1 and 2 repeatedly peptide basic skeleton _{NH 2 -E (OtBu) -AR (} Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin).

4) Cleavage: Cleavage cocktail was added to the synthesized peptide resin to separate the peptide from the resin.

5) Cooling diethyl ether was added to the resulting mixture, and the resulting peptide was precipitated by centrifugation.

6) After purification by Prep-HPLC, molecular weight was confirmed by LC / MS and frozen to powder.

Example 2: Effect of PEP1 on differentiation of MDSC

Inhibitory Effect of PEP1 on Marker Expression Indicating Maturation of MDSC

Experimental animals and target cells, peptides

(PMN-MDSC) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) isolated from the spleen of mice with solid tumors to analyze the maturation of undifferentiated myeloid cells (CD11c, F4 / 80, MHC class II) of MDSC when treated with PEP1 in the presence of monocytic myeloid-derived suppressor cells (Mo-MDSC) Expression was observed. PMN-MDSC and Mo-MDSC were isolated from the spleen of 4.5-week-old Balb / c mice transplanted with CT26 cancer cells for 3 days under GM-CSF. The results were compared with the results of EL4 cancer cell transplanted 3.5-week C57BL / 6 The mouse spleen was separated and cultured for 3 days under GM-CSF. The results of the experiment were collected and the average value was determined for each marker. PEP1 was prepared by the method in Example 1 and a control peptide was prepared by the method of Example 1 using a scrambled peptide having the same 16 amino acids as PEP1 and differing only in the order of the amino acid sequence, Were used.

Method of analyzing expression of marker

MDSC maturation markers in the target cells were measured for fluorescence by fluorescence activated cell sorting (FACS). The degree of expression of the markers was measured by fluorescence and the fluorescence values measured by PEP1 (GV1001), control peptide, no peptide, and isotype control were measured for each marker. (MFI, Mean Fluorescence Intensity) were analyzed (see Figs. 1 to 6). The average fluorescence values were analyzed by separating them into raw data and relative values. Relative values were expressed in a way that indicates the relative ratios of the different groups by setting the isotype treatment group to 1. Markers in MDSC maturation differ in the factors regulated by PEP1 by type (PMN- and Mo-) of MDSC, CD11c and F4 / 80 in PMN-MDSC type and MHC II in Mo-MDSC type respectively Respectively.

Analysis

Analysis of the mean fluorescence values revealed that the expression of MDSC markers was more suppressed in the PEP1-treated group than in the control peptide in the PMN-MDSC type for CD11c and F4 / 80 in the absolute value (see FIGS. 1 and 2 ). In the case of absolute values, the expression of MHC II was also inhibited in the PEP1-treated group in the Mo-MDSC type than in the control group (see FIG. 3). In order to compare the tendency of the change in various groups, the expression of the mean fluorescence value as the relative value was also suppressed in the PEP1-treated group as compared with the control group, as in the case of the absolute value analysis (see FIGS. 4 to 6 ).

Example 3: Inhibitory effect of MDSC upon combination administration of PEP1 and conventional cancer drugs

In order to investigate the inhibitory effect of MDSC upon combination administration of PEP1 and conventional cancer drugs, 24 healthy control subjects (Comparative Example 1) and 40 pancreatic cancer patients whose age and gender matched each other (Comparative Example 2, Example 3 ) Were sampled from 20 to 30 ml of venous blood. The control group was recruited from a surgical minor operation clinic at the Royal Surrey Country Hospital in England. All 40 patients have never had autoimmune disease or recent steroid treatment, and the control group has never had cancer. All subjects participating in the clinical trial submitted a written consent and were approved by the local human inspectors human investigators committee. All patients received gemcitabine plus capecitabine (GemCap). Forty patients were divided into two groups, arm2 (Comparative Example 2) and arm3 (Example 3). Arm2 (Comparative Example 2) was 19 persons, and arm 3 (Example 3) was 21 persons. Arm2 (Comparative Example 2) was administered concomitantly with gemcitabine and capecitabine, while arm3 (Example 3) was administered simultaneously with gemcitabine and capecitabine plus PEP1 peptide and its adjuvant, GM-CSF.

- Comparative Example 1 (Control): 24 healthy controls, gemcitabine-capecitabine (GemCap) administration

- Comparative Example 2 (arm2): 19 pancreatic cancer patients, gemcitabine-capecitabine (GemCap) administration

Example 3 (arm3): 21 pancreatic cancer patients, gemcitabine-capecitabine (GemCap) + PEP1-GM-CSF administration

Gemcitabine (1,000 mg / m ² ) was administered intravenously once a week for 4 weeks, 1 week for the first 3 weeks, and 1 week for the first 3 weeks. At the same time, capecitabine was orally administered twice a day for three weeks and not administered for one week. The dosage 1,660mg / m ² / day was (830mg / m ² 1 twice daily). MDSC orbits of 19 patients with arm 2 were analyzed during GemCap treatment and after two cycles of GemCap treatment. MDSC trajectories were also analyzed in 21 patients with arm3 treated with GemCap and PEP1. The primary PEP1 was administered intravenously at week 1, week 2, week 3, week 4, week 6 and week 10, 0.56 mg of PEP 1 at day 1, 3, and 5, -CSF was administered intradermally at the same site as PEP1 with an adjuvant of PEP1.

Peripheral blood samples were collected before and after GemCap administration. Arm2 patients receiving gemcitabine plus capecitabine alone received blood samples after 7 weeks of treatment, before the sixth gemcitabine dose, and when taking capecitabine. After 10 weeks of treatment, immediately after 7th gemcitabine administration and 8th gemcitabine and capecitabine were given to coincide with the immunomonitoring time of arm3 patients receiving gemcitabine, capecitabine and PEP1 When a blood sample was taken.

Three patients in arm 3 received blood at week 14 and two patients at week 18, consistent with the time of PEP1 administration. The collected blood samples were transferred to a li-heparin tube (BD Biosciencs, Europe) or a CPT tube and transferred to a Biomarker repository located in the Liverpool Cancer Trial Unit, UK. PBMCs were separated using a Ficoll-Hypaque gradient, counted, and frozen at minus 80 ° C for later analysis and stored in liquid nitrogen.

Immunophenotypic analysis of cells

Peripherl blood mononuclear blood cells were recovered using a 0.15 M phosphate-buffed saline solution (Dulbecco's A) (Oxoid, UK). Partial samples of the recovered cells were used for MDSC analysis and survival and dead cells were distinguished using the LIVE / DEAD Cell Stain kit (Invitrogen, UK). Anti-HLA-DR-APC-Cy7, anti-Lin1 (CD3,14,16,19,20,56) -FITC and anti-CD1b-PECy7 after cell washing with binding buffer solution (BD Biosciences, Europe) The anti-human monoclonal antibody was used for flow cytometry analysis. After immunostaining, the cells were washed with a binding buffer solution and analyzed using a MACSQuant flow cytometer using MACSQuantify software (Miltenyi Biotec).

Delayed-type hypersensitivity (DTH) skin tests

100 μg of PEP1 was injected intracutaneously on the opposite side of the belly vaccine site. Patients measured the size of the DTH response 48 hours after intradermal injection and were asked to report to the physician. The DTH reaction was accompanied by erythema and curing, and was shown to have an average diameter of 5 mm.

In vitro proliferation assays

The thawed PBMCs were dispensed into a 48-well plate (ThermoFisher Scientific, USA) to a concentration of 2x10 ⁶ cells per well, and then incubated with 10% pooled human serum (Innovative Research, USA), X-VIVO containing 20 ug / ml PEP1 peptide , UK) for 3 days. After incubation, 10 units / ml IL-2 (Peprotech, UK) was added to the medium. On day 11 of culturing, PEP1-enriched cells were harvested and then 1x10 ⁵ cells / well were placed in a round-bottom 96-well plate. Pre-stimulated cells were allowed to act as antigen-presenting cells by adding irradiated (45 Gy) autologous PBMCs to give 50 μl per well of 1 × 10 ⁵ cells. Cell proliferation specific for PEP1 was determined in 100 μl of control medium (PEP1 (20 μg / ml) or a positive control (5 μg / ml) was added to the cells for two days and analyzed after the cells were cultured and 1 μCi of ³ H-thymidine was added for 16 hours per well before counting the cells. ) Was defined as a positive proliferative response to PEP1 when the stimulation index (SI) was 1.8 or more than the difference in the number of cells per minute.

Cytokine Analysis

The cytokine levels of patient serum collected during PBMC collection were analyzed using the BioRad BioPlex 27 Assay from BioRad BioPlex Instrument.

Tumor Burden assessment

Individual analyzes were performed on chemotherapy pre- and post-tumor tumors using PRECIST V1.1 CRITERIA for lesion measurement via CT scan. Tumor burden (mm) was measured using the sum of long axis measurements of tumor lesions and short axis measurements of pathologic lymph.

Statistical Analysis

Welch's correction and unpaired t-test were used to compare the median values of Lin-DR-CD11b + cells in advanced pancreatic cancer patients and controls. Spearman's rank test was used to analyze the association of MDSC baseline values with cytokine baseline values and the differences between the cytokines and the MDSC median values were analyzed using the nonparametric Mann-Withney test. Paired Wilcoxon test was used to compare cytokine levels after chemoprevention.

To calculate the absolute value of the MDSC difference, the MDSC value before treatment was subtracted from the MDSC value after treatment, and the absolute value of the MDSC difference was calculated as the absolute value of the MDSC difference by dividing the absolute value of the MDSC change by the value measured before the treatment. The calculated data are distributed asymmetrically and graphically on a log scale, but all analyzes performed represent the original dimensions using a nonparametric approach. Wilcoxon signed rantks test was used to analyze differences within each treatment group. Wilcoxon two-sample tests were used to compare the pretreatment MDSC values, the MDSC values, the MDSC difference absolute values, and the MDSC difference% absolute values of arm 2 and arm 3 treatment groups. Wilcoxon two-sample tests were used to compare the absolute values of percentages of MDSC difference between patients with disease control (PR, SD) and those with progressive disease (PD). For trend consistency, the sensitivity analysis includes data reanalyzed for disease-suppressing patients to eliminate effects due to tumor size changes and data reanalyzed for arm3 after 10 weeks of post-treatment.

Analysis

1. Association of MDSC with proinflammatory cytokine levels

As shown in Fig. 1, there was no correlation between the level of proinflammatory cytokine and the reference value of Lin-DR-CD11b + cells in pancreatic cancer patients.

We analyzed cryopreserved peripheral blood mononuclear cells (PBMCS) from 40 advanced pancreatic cancer patients who received gemcitabine and capecitabine. Twenty-one patients out of 40 received the telomerase vaccine PEP1, gemcitabine, and capecitabine. The pre-treatment baseline values of 21 patients were used to calculate the association of pretreatment median levels with inflammatory cytokine MDSC levels. To analyze the effects of gemcitabine and capecitabine on Lin-DR-CD11b + cells, we analyzed samples of 19 patients who received only consecutive gemcitabine and capecitabine. The phenotypes obtained from the analyzes were used to label MDSCs based on the results of Kotsakis.

The number of Lin-DR-CD11b + cells in 40 pancreatic cancer patients was increased compared to the control (Comparative Example 1) ( p <0.0001). The median value of Lin-DR-CD11b + cells in the patient group was 1.85 (range 0.62-845), and the median cell value of the corresponding 24 control group was 0.82 (range 0.16-0.22).

In 33 pancreatic cancer patients with all cytokine data (Speraman's coefficient: IL-6 = 0.153, IL-1β = 0.22, VEGF = -0.0389, TNFa = 0.0587, MCP-1 = -0.226), inflammatory cytokines And MDSC reference values. When the MDSC level was differentiated from the median value, a significant difference in the baseline value of the cytokine between the low MDSC level and the high level person could not be confirmed (FIG. 1).

2. Results of Comparative Example 2 (arm2) in which only GemCap was administered

The results of Comparative Example 2 (arm2) in which only GemCap was administered showed that the number of Lin-DR-CD11b + cells was decreased in some cases, but not by GemCap, but by the degree of disease control or cancer-related inflammation. Specifically, it is as follows.

Tumor volume Eight out of 19 patients treated with gemcitabine plus capecitabine (comparative example 2, arm 2) had a reduced number of Lin-DR-CD11b + cells. Five of the seven progressive disease (PD) patients with cancer progressed to increased levels of Lin-DR-CD11b + cells and two levels decreased (range -60 to +662%). Six of 10 patients with Stable disease (SD) had elevated levels of Lin-DR-CD11b + cells and 4 patients (range, -68 to + 604%). Patients who had partial response to the Pep1 vaccine had decreased Lin-DR-CD11b +%. The size of the tumor was precisely measured and no increase or decrease in the sum of the longest diameter of the significant tumors was observed in eight of the 10 patients who were not ongoing. The direct effect of tumor volume change on Lin-DR-CD11b + cells was not significant. Lin-DR-CD11b +% increased in 5 patients and Lin-DR-CD11b +% decreased in 3 patients. These data indicate that Lin-DR-CD11b + is not reduced by gemcitabine and capecitabine itself. Changes in Lin-DR-CD11b +% showed a tendency to follow the tumor response. This was demonstrated in patients with a Lin-DR-CD11b + baseline greater than the median, and a reduction in MDSC would be of immune benefit to this patient group (Table 3).

Six of the patients with a Lin-DR-CD11b +% threshold greater than the median increased Lin-DR-CD11b +%, and 3 had no volume change (0%). One of the three patients with reduced Lin-DR-CD11b + showed partial response and a volume change of 11%. Of these, only Lin-DR-CD11b +% was lower than the median after administration of gemcitabine plus capecitabine. When Arm2 and arm3 were combined, the absolute value of MDSC change was associated with response to anticancer drugs ( p = 0.02), and the MDSC levels of 9 patients with cancer progression were increased (median = 0.47) (Median = -0.49) of the 31 patients were decreased.

During the treatment, other inflammatory cytokines and IL-6 were used to analyze the degree of cancer-related inflammation and MDSC levels. The results are shown in Table 4.

We hypothesized that cancer-associated inflammation, which induces persistent MDSC production, is induced in patients with increased MDSC%, although there is little change in tumor volume. IL-6 levels were elevated in 7 out of 19 patients with Arm2 during gemcitabine and capecitabine, and 4 of them had cancer. MDSC% of the 3 patients who had not undergone cancer increased. Table 4 shows the changes in MDSC% for inflammatory cytokines during the administration of gemcitabine and capecitabine in 10 of 19 patients whose cancer is not ongoing. IL-6 levels in one of the patients (patient 8) decreased from 152.72 to 8.66 pg / ml during the 7-week chemotherapy regimen, This was correlated with MDSC%, which decreased from 2.54 to 1.59. The MDSC level baseline was lower than the median and was lower than the median after gemcitabine and capecitabine treatment. Two patients with a MDSC baseline greater than the median increased MDSC levels continuously during treatment and a significant increase in IL-6 levels (Patients 1 and 3). The Vascular Endothelial Growth Factor of Patient 3 also increased from 37.59 to 70.69 pg / ml, which was the only increase in VEGF levels among patients without cancer progression.

3. Comparison of the number of MDSCs of Comparative Example 2 (arm2) and Example 3 (arm3)

Gemcitabine-capecitabine (GemCap) and PEP1-GM-CSF were administered to 19 pancreatic cancer patients in comparison 2 (arm2) and 21 pancreatic cancer patients treated with gemcitabine-capecitabine (GemCap) + PEP1-GM-CSF The results of comparing the number of MDSCs according to Example 3 (arm 3) are shown in FIG. 8 and Table 5.

MDSC changes were analyzed and compared between patients receiving gemcitabine and capecitabine (GemCap) alone (comparative example 2, arm2) and patients receiving GemCap and PEP1 (example 3, arm3). The summary statistics comparing the MDSC after administration of Arm2 and arm3 before administration are shown in Table 5, and the MDSC change values are logarithmically shown in FIG. Before treatment MDSC value arm3 (Example 3), even though higher than the arm 2 (Comparative Example 2) and (p = 0.08), arm 3 ( Example 3) in patients with MDSC level ((p = 0.007 and p = 0.006 There was no significant decrease in arm 2 ( p = 0.60 and p = 0.62 for absolute and percentage change) in arm 2 ( p = 0.60 and p = 0.62 for absolute and percentage change) Arm 2 and arm 3 were statistically significant (p = 0.04 and p = 0.06 for absolute and percentage change) in changes in MDSC levels, while the MDSC values (P> 0.99) There was a significant difference between arm 2 and arm 3 patients, but consistency was maintained after 10 weeks of treatment with SD subgroups. When the number of patients with reduced MDSC% was compared, arm 3 (Example 3) (N = 19), whereas in arm 2 (n = 2) only 8 of the 19 patients It showed a cow.

A positive proliferation assay (PEP1) was performed in 21 arm 3 patients receiving GemCap and PEP1. Nine out of 21 patients showed immune response, and 8 of 9 patients had decreased MDSC% during treatment. The baseline value of Lin-DR-CD11b +% in 6 of 9 patients was higher than the median value of patients, and the MDSC level decreased.

All patients with immune response were controlled for radiation disease during blood sampling to analyze the proliferative response in the same way as MDSC analysis.

From the results of Example 2 and Example 3, it was confirmed that the effect of inhibiting the differentiation of MDSC cells in cancer-induced experimental animals when PEP1 alone was administered (Example 2) , The combination of PEP1 and the conventional anticancer agent (GemCap) was able to confirm the MDSC inhibitory effect of the cancer patients through the related cytokine (Example 3). Therefore, it can be seen that PEP1 has an effect on the inhibition of MDSC, and it can be seen that there is a possibility that it can be used for the composition for inhibiting MDSC containing PEP1 and its treatment using therewith.

<110> KAEL-GEMVAX CO., LTD.          KIM, Sang Jae <120> Composition for inhibiting myeloid-derived suppressor cells <130> OF14P080 / PCT &Lt; 150 > PCT / KR2014 / 004760 <151> 2014-05-28 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys   1 5 10 15 <210> 2 <211> 1132 <212> PRT <213> Homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser   1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly              20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg          35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro      50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu  65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val                  85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro             100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr         115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val     130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr                 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro His Ala Ser Gly             180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg         195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg     210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp                 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val             260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala         275 280 285 Leu Ser Gly Thr Arg His Ser Ser Ser Val Gly Arg Gln His His     290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly                 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro             340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser         355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln     370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg                 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln             420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu         435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe     450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser                 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met             500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys         515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe     530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr                 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His             580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln         595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile     610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser                 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg             660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg         675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro     690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln                 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His             740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp         755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser     770 775 780 Pro Leu Arg Asp Ala Val Valle Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His                 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro             820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp         835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu     850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys                 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu             900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe         915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser     930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly                 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn             980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln         995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln    1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu                1045 1050 1055 Gly Ala Lys Gly Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp            1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr        1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser    1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp                1125 1130

Claims (31)

As a composition for inhibiting myeloid-derived suppressor cells (hereinafter "MDSC"),
The composition comprises a peptide comprising SEQ ID NO: 1 as an active ingredient for inhibiting MDSC, a peptide having a sequence homology of 80% or more with the peptide sequence, or a fragment thereof, wherein the peptide is an amount effective to inhibit MDSC &Lt; / RTI &gt; 2. The composition according to claim 1, wherein said composition is for administration to a subject in need of inhibiting MDSC. The composition for inhibiting MDSC according to claim 1, wherein the bone marrow-derived inhibitory cell is a bone marrow-derived inhibitory cell of an individual having a tumor. 4. The method of claim 3, wherein the tumor is selected from renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, lung cancer, blood cancer, prostate cancer, adenocarcinoma, prostate cancer, and pancreatic cancer. Composition for inhibiting MDSC. The composition for inhibiting MDSC according to claim 1 or 3, wherein the composition is administered in combination with an anticancer agent. The composition for inhibiting MDSC according to claim 5, wherein the anticancer agent is a chemotherapeutic agent, and the chemotherapeutic agent is at least one of deoxynucleoside analogue and fluoropyrimidine. The method according to claim 6,
Wherein the deoxynucleoside analog is gemcitabine, and the fluoropyrimidine is 5-fluorouracil or capecitabine. 7. The composition according to claim 5, wherein the composition is administered in combination with an adjuvant. 9. The composition for inhibiting MDSC according to claim 8, wherein the adjuvant is a cytokine adjuvant. 10. The composition for inhibiting MDSC according to claim 9, wherein the cytokine adjuvant is granulocyte-macrophage colony-stimulating factor (GM-CSF). 11. The composition for inhibiting MDSC according to any one of claims 1 to 10, wherein the MDSC is Lin-DR-CD11b +. 11. A composition for inhibiting MDSC according to any one of claims 1 to 10; And an MDSC inhibitor kit containing instructions. 13. The kit according to claim 12,
A kit for inhibiting MDSC further comprising an anticancer agent. 14. The MDSC inhibitor according to claim 13, wherein the anticancer agent is a chemotherapeutic agent, and the chemotherapeutic agent is at least one of a deoxynucleoside analogue and a fluoropyrimidine. 15. The method of claim 14,
Wherein the deoxynucleoside analog is gemcitabine and the fluoropyrimidine is 5-fluorouracil or capecitabine. 13. The kit according to claim 12, wherein the kit further comprises an adjuvant. 17. The kit for inhibiting MDSC according to claim 16, wherein the adjuvant is a cytokine adjuvant. 18. The kit for inhibiting MDSC according to claim 17, wherein the adjuvant is a granulocyte-macrophage colony-stimulating factor (GM-CSF). 13. The MDSC inhibitor kit according to claim 12, wherein said instructions include administration of said MDSC inhibiting composition, said anticancer agent and / or said adjuvant in combination. 13. The MDSC inhibitor kit according to claim 12, wherein the MDSC inhibitory kit is to ameliorate, prevent or treat a disease or symptom of MDSC through MDSC inhibition. 13. The kit for inhibiting MDSC according to claim 12, wherein said MDSC inhibiting kit is for improving, preventing or treating cancer through inhibition of MDSC. 22. The method of claim 21, wherein said cancer is selected from renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, lung cancer, blood cancer, prostate cancer, adenocarcinoma, prostate cancer, and pancreatic cancer. MDSC Inhibition Kit. As a method for inhibiting myeloid-derived suppressor cells ("MDSC"),
MDSC inhibition comprising administering to a subject in need of MDSC inhibition an effective amount of a peptide comprising SEQ ID NO: 1 effective to inhibit MDSC, a peptide having 80% or more sequence homology with said peptide sequence, or a fragment thereof, Way. A peptide comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof;
Anticancer agents; And
A composition for inhibiting MDSC comprising an adjuvant. A peptide comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof;
Anticancer agents; And
An adjuvant. A peptide comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof;
Anticancer agents; And
Adjuvant. &Lt; / RTI &gt; 27. The method according to any one of claims 24 to 26,
Wherein the anticancer agent is a chemotherapeutic agent, the chemotherapeutic agent is at least one of a deoxynucleoside analogue and a fluoropyrimidine,
Wherein the adjuvant is a cytokine adjuvant. 28. The method of claim 27,
Wherein said peptide is a peptide consisting of SEQ ID NO: 1,
Wherein the deoxynucleoside analog is gemcitabine, the fluoropyrimidine is 5-fluorouracil or capecitabine,
Wherein the adjuvant is GM-CSF. A composition comprising a peptide comprising SEQ ID NO: 1, a peptide having 80% or more sequence homology with the peptide sequence, or a peptide thereof;
Anticancer agents; And
An anti-cancer kit containing instructions. A method for improving, treating or preventing cancer,
An effective amount of a peptide comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof, in combination with an anticancer agent and / or adjuvant, A method of improving, treating or preventing cancer, comprising administering to a subject in need thereof. A peptide comprising SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the above peptide sequence, or a fragment thereof (hereinafter, referred to as " peptide ") for producing a composition for inhibiting myeloid-derived suppressor cells The use of the peptides.

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