WO2015182798A1 - Composition for inhibiting myeloid-derived suppressor cells (mdscs) - Google Patents

Abstract

Disclosed is a novel use of a peptide including SEQ ID NO: 1, a peptide having at least 80% sequence identity to the peptide sequence, or a peptide as a fragment thereof. Specifically, disclosed is a use of the peptide for inhibiting myeloid-derived suppressor cells (MDSCs). The suppression problem of the immune response by MDSC can be solved by inhibiting MDSC. The peptide is used in combination with other anticancer agents and adjuvants, thereby effectively inhibiting MDSC, and thus can relieve, treat, or prevent MDSC-related diseases or symptoms. Furthermore, provided can be a vaccine having no side effects associated with MDSC, especially, a cancer vaccine, an anticancer kit, or an anticancer method.

Description

Bone Marrow-Derived Suppressor Cells (MYELOID-DERIVED SUPPRESSOR CELLS, MDSCS)

In the present specification, a composition for inhibiting myeloid-derived suppressor cells (MDSCs), a kit for inhibiting MDSC, a method for inhibiting MDSC, a composition for anticancer, a kit for anticancer, an anticancer method, or a novel use of telomerase peptide It is about.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of immature bone marrow cells that have had differentiation programs stagnated by various tumor-secreting factors. MDSCs inhibit the activity of cytotoxic T lymphocytes in a variety of ways.

The function of MDSC in vivo is, in part, to protect the body from the harmful effects of excessive immune stimulation in acute and chronic infections and to limit the development of autoimmune responses to tissue antigens released by trauma. As such, while the action of MDSCs inhibits the autoimmune response, the proliferation of MDSCs is also associated with a pathological point of view. Activation is mediated through several transcription factors, resulting in 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 MDSC allows the immunosuppressive environment to be sustained, which in turn exposes the living body to allergen and / or viral infections, which can lead to chronic inflammation. Chronic inflammation can lead to tissue damage if the body is unable to respond effectively.

The 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 of the spleen in tumor-induced mice [Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM (2005) Gemcitabine eliminate 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 reported to be greater than gemcitabine.

Another method for inhibiting MDSC is to add miR-142 and / or miR-223 ribonucleotides to MDSC (WO 2013/082591). miR-142 and / or miR-223 ribonucleotides can be reduced in number by allowing MDSCs to differentiate into macrophages, dendritic cells, and the like.

As another method for inhibiting MDSC, a method using bisphosphonates or CCR2 inhibitors as adjuvant is known (WO 2011/116299). Bisphosphonates include clodronate, zoleronate, palmidronate, ethidronate or other bisphosphonate drugs. CCR2 inhibitors include RS 1028595 or PF-04178903.

Meanwhile, granulocyte-macrophage colony-stimulating factor (GM-CSF), which is added as an adjuvant for vaccines in all clinical trials, is known to increase MDSC in the tumor micro-environment [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, low doses of GM-CSF as a vaccine adjuvant are known to increase 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 of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553.

[Preceding technical literature]

[Patent Documents]

(1) WO 2013/082591

(2) WO 2011/116299

[Non-Patent Documents]

(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, Rivoltini L et al (2007) Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553

In one aspect, the 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 inhibited by MDSC.

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

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

In another aspect, an object of the present invention is to alleviate, treat or prevent a disease or condition associated with MDSC.

In another aspect, it is an object of the present invention to provide an anticancer composition, anticancer kit or anticancer method that is free of the 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 without the side effects of lowering the immune response according to MDSC.

In one aspect, the present invention is a composition for inhibiting myeloid-derived suppressor cells (MDSCs), the composition is a peptide comprising SEQ ID NO: 1 as an active ingredient for MDSC inhibition, the peptide sequence and 80% Peptide having a sequence homology, or a fragment thereof, the peptide is a composition for inhibiting myeloid-derived suppressor cells (MDSCs), contained in an amount effective for MDSC inhibition.

In another aspect, the invention provides a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And it may be a composition for inhibiting MDSC containing an adjuvant.

In another aspect, the invention provides a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And it may be an anticancer composition comprising an adjuvant.

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

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

In one aspect, the kit for inhibiting MDSC may be to improve, prevent or treat a disease or symptom caused by MDSC through MDSC inhibition.

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

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

In one aspect, the present invention provides a method for inhibiting myeloid-derived suppressor cells (MDSCs), an effective amount of a peptide comprising SEQ ID NO: 1, the peptide sequence and at least 80% sequence Or a peptide that is homologous, or a fragment thereof, to a subject in need of MDSC inhibition. In one aspect, the method may further comprise administering an anticancer 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 for improving, treating or preventing cancer, wherein the peptide comprises an effective amount of a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof. And in combination with an anticancer agent and / or adjuvant, may be a method for cancer improvement, treatment or prevention comprising administering to a subject in need of improvement, treatment or prevention of cancer.

In one aspect, the present invention, to prepare a composition for inhibiting myeloid-derived suppressor cells (MDSCs), a peptide comprising SEQ ID NO: 1, having a sequence homology of 80% or more of the peptide sequence Peptides, or fragments thereof, may be used.

In one aspect, the present invention may inhibit MDSC.

In another aspect, the present invention may activate immune.

In another aspect, the present invention can activate immunity 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 a cancer vaccine is inhibited by MDSC.

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

In another aspect, the present invention may provide an anticancer composition, an anticancer kit, or an anticancer method that does not have the side effects of lowering the immune response according to MDSC.

In another aspect, the present invention can provide a cancer vaccine that is free of the side effects of lowering the immune response following MDSC.

1 is a graph showing the relationship between MDSC and the levels of proinflammatory cytokine. Comparison of the proinflammatory cytokines of patients with high and low levels of MDSC showed no correlation between the levels of MDSC and proinflammatory cytokine.

Figure 2 is a log after analyzing and comparing the MDSC changes in the patient group (comparative example 2, arm2) that was administered only in combination with gemcitabine and capecitabine, and the patient group (Example 2, arm3) that was simultaneously administered GemCap and pep1 It is a graph in scale.

All known references mentioned herein are incorporated in their entirety as part of this specification.

In the present specification, "MDSC inhibition" is a concept that includes not only reducing the number of MDSCs but also inhibiting the activity of MDSCs. Reducing the number includes not only inhibiting the production of cells, but also killing or differentiating cells that have already been formed. In addition, all mechanisms that are referred to as "inhibition" from a biological standpoint are included.

As used herein, “combined administration” or “combined administration” refers not only to simultaneous administration in terms of physical time, but also to two or more drugs in combination, according to the unique schedule of each drug being combined as in conventional combination therapy. Administering.

We have found a significant increase in MDSCs in cancer patients. This means that MDSCs can inhibit the effectiveness of cancer vaccines administered for the treatment of cancer patients, and as a result can render cancer treatment impossible. For cancer treatment with cancer vaccines, it is necessary to reduce the increased MDSC in cancer patients.

In addition, the present inventors used a combination of two chemotherapeutic agents, gemcitabine, and 5-fluorouracil (5-FU) or capecitabine, which are known to directly reduce MDSC. It has been found that, unlike single use, it does not reduce MDSC. Rather, a significant number of patients showed elevated MDSCs. This may act as a big limitation of the combination of gemcitabine and 5-fluorouracil (or capecitabine).

Using the peptides disclosed herein can significantly reduce MDSC. Reducing MDSCs can solve a series of pathological problems associated with MDSCs.

The peptides disclosed herein may be some fragments or analogs of telomerase. Telomere is a genetic material repeatedly present at the end of a chromosome and is known to prevent damage to the chromosome or binding to another chromosome. Each time a cell divides, the telomeres become slightly shorter. After a certain number of cell divisions, the telomeres become very short, and the cells stop dividing and die. On the other hand, elongation of telomeres is known to prolong cell life. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening, so that cancer cells can continue to proliferate without dying.

The peptides disclosed herein are peptides having a sequence of SEQ ID NO: 1 or a peptide that is a fragment of sequence SEQ ID NO: 1, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, Peptides having at least 98%, at least 99% sequence homology.

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

The peptides disclosed herein may include peptides having sequence homology with a peptide comprising SEQ ID NO: 1 or a fragment thereof. In addition, the peptides disclosed herein, peptides or fragments thereof comprising SEQ ID NO: 1 and 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, six or more amino acids Or peptides with seven or more amino acids changed.

In one aspect of the invention, the peptide may consist of up to 30 amino acids.

The peptide described in SEQ ID NO: 1 is shown in Table 1 below. The "name" in Table 1 below is to distinguish peptides. In one aspect of the invention, the peptide set forth in 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 that is a fragment of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence corresponds to a peptide included in telomerase. And "synthetic peptides" selected and synthesized at the positional peptides. SEQ ID 2 shows the amino acid sequence of the entire telomerase.

Table 1

In one aspect of the invention, amino acid changes belong to a property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

As used herein, "amino acid" includes not only the 22 standard amino acids that are naturally incorporated into the peptide, but also D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. Meanwhile, in another aspect of the present invention, the peptide may include a non-standard amino acid or the like which has been post-translational modified. 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 (e.g., beta-elimination deimidization) , Deamidation) and structural changes (eg, formation of disulfide bridges). It also includes changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that occur during the linkage with crosslinkers to form peptide conjugates.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, a 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 that is SEQ ID NO: 1 or a fragment thereof. Amino acid changes in the wild type polypeptide as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein. Examples of conservative substitutions include 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), aromatic amino acids (phenylalanine, Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, 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.

TABLE 2

Original amino acid	Exemplary residue substitutions	Preferred residue substitution
Ala (A)	val; leu; ile	Val
Arg (R)	lys; gln; asn	Lys
Asn (N)	gln; his; asp, lys; arg	Gln
Asp (D)	glu; asn	Glu
Cys (C)	ser; ala	Ser
Gln (Q)	asn; glu	Asn
Glu (E)	asp; gln	Asp
Gly (G)	Ala	Ala
His (H)	asn; gln; lys; arg	Arg
Ile (I)	leu; val; met; ala; phe; norleucine	Leu
Leu (L)	norleucine; ile; val; met; ala; phe	Ile
Lys (K)	arg; gln; asn	Arg
Met (M)	leu; phe; ile	Leu
Phe (F)	leu; val; ile; ala; tyr	Tyr
Pro (P)	Ala	Ala
Ser (S)	thr	Thr
Thr (T)	Ser	Ser
Trp (W)	tyr; phe	Tyr
Tyr (Y)	trp; phe; thr; ser	Phe
Val (V)	ile; leu; met; phe; ala; norleucine	Leu

In one aspect, the present invention is a composition for inhibiting myeloid-derived suppressor cells (MDSCs), the composition is a peptide comprising SEQ ID NO: 1 as an active ingredient for MDSC inhibition, the peptide sequence and 80% Peptide having a sequence homology, or a fragment thereof, the peptide is a composition for inhibiting myeloid-derived suppressor cells (MDSCs), contained in an amount effective for MDSC inhibition.

In another aspect, the invention provides a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And it may be a composition for inhibiting MDSC containing an adjuvant.

In another aspect, the invention provides a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof; Anticancer agents; And it may be an anticancer composition comprising an adjuvant.

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

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

In one aspect, the kit for inhibiting MDSC may be to improve, prevent or treat a disease or symptom caused by MDSC through MDSC inhibition.

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

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

In the above-mentioned kits, the instructions may include a description of how the composition for inhibiting MDSC containing the peptide is used in combination with an anticancer agent. Specifically, it may include a description of the dosage, administration time, administration method and the like of each agent. In addition, the instructions may include uses for inhibiting MDSC, anticancer use or cancer vaccine use. Specifically, all diseases or symptoms that need to inhibit MDSC can be included. In addition, the instructions may include side effects and precautions associated with the administration.

In one aspect, the present invention provides a method for inhibiting myeloid-derived suppressor cells (MDSCs), an effective amount of a peptide comprising SEQ ID NO: 1, the peptide sequence and at least 80% sequence Or a peptide that is homologous, or a fragment thereof, to a subject in need of MDSC inhibition. In one aspect, the method may further comprise administering an anticancer 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 for improving, treating or preventing cancer, wherein the peptide comprises an effective amount of a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof. And in combination with an anticancer agent and / or adjuvant, may be a method for cancer improvement, treatment or prevention comprising administering to a subject in need of improvement, treatment or prevention of cancer.

In one aspect, the cancer is not particularly limited, but may be 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, to prepare a composition for inhibiting myeloid-derived suppressor cells (MDSCs), a peptide comprising SEQ ID NO: 1, having a sequence homology of 80% or more of the peptide sequence Peptides, or fragments thereof, may be used.

In the present specification, bone marrow-derived suppressor cells (MDSC) may be bone marrow-derived suppressor cells of an individual having a tumor. In addition, the composition may be for administration to a subject in need of inhibition of MDSC. MDSC functions to suppress immunity by inhibiting the activity of cytotoxic T lymphocytes. There is a net function of suppressing unnecessary and excessive immune responses, such as autoimmunity, but there is also a reverse function of inhibiting immunity in situations where an immune response is needed, causing or worsening disease, or impeding proper treatment. For example, MDSC is much increased in tumor or cancer patients, which negates the efficacy of cancer vaccines by significantly reducing the effects of cancer vaccine administration. In this situation, effectively reducing the number of MDSCs will enable smooth and effective cancer treatment.

Diseases or symptoms caused by MDSC herein are apparent in the art. Diseases or symptoms caused by MDSC referred to herein include all diseases or symptoms caused by MDSC known in the art. Bone diseases such as, for example, 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; Autoimmune diseases such as intraocular autoimmune diseases; Inflammatory bowel disease, Cancer cachexia; Or tuberculosis (TB), but is not limited thereto.

MDSC in this specification may include all MDSCs regardless of their phenotype. Various phenotypes of MDSC are known in the art. For example, the phenotype is one 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 / killing identifiers. Can be. For example, phenotypes include MDSC1 based on the labels IL4Ra + and CD14 +; MDSC2 based on the labels IL4Ra + and CD15 +; MDSC1 and MDSC2 based on the label IL4Ra +; MDSC3 based on the labels 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 labels 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 +, and the like. For example, the phenotype can be Lin-DR-CD11b +.

The concentration of peptides in the compositions disclosed herein can be routinely determined as known in the art. For example, the composition according to one aspect of the present invention comprises 10 mg / L to 1000 peptides comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof. It may be included in the content of mg / L, specifically 10 mg / L to 500 mg / L, more specifically 30 mg / L to 200 mg / L, but when the difference in effect depending on the dose can be adjusted appropriately. When included in the above range or less, it is not only appropriate to exhibit the intended effect of the present invention, but also satisfies both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness. .

Since the dosage, administration method, administration cycle, and the like of the peptide herein are well known in the art, it may be administered according to the standards known in the art according to the condition of each patient. Specific dosage determinations are within the level of those skilled in the art, and their daily dosage may be, for example, specifically 1 μg / kg / day to 10 g / kg / day, more specifically 10 μg / kg / day to 100 mg / kg / day, and more specifically, 50 ㎍ / kg / day to 10 mg / kg / day, but is not limited to this, depending on a variety of factors, such as age, health conditions, complications of the subject to be administered Can be. For example, the peptide can be administered via intradermal administration. Dosage intervals may be administered once daily at two-day intervals and may be further extended over time. For example, after administration in weeks 1, 2, 3, and 4, the intervals may be spaced into weeks 6 and 10. The dosage may be 0.1 to 3 mg once administered on an adult basis. The dosage can 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. In addition, the dosage 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, 0.6 mg or less.

In one aspect, the peptides described herein can be administered in combination with an anticancer agent. Anticancer agents include, but are not limited to, chemotherapeutic and biotherapeutic agents. In particular, it may 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. Metabolism inhibitors refer to groups of molecules that interfere with DNA and RNA synthesis. Most metabolism inhibitors have a structure similar to the building blocks of DNA and RNA. Subtypes of metabolic antagonists include, but are not limited to, folate antagonists (anti-folates), fluoropyrimidines, deoxynucleoside analogues and thiopurines. Fluoropyrimidines include, but are not limited to, fluorouracil and capecitabine. Capecitabine is a prodrug of 5-fluorouracil. Deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, vidaza, fludarabine, nelarabine, cladribine Cladribine, clofarabine and pentostatin, but are not limited thereto. The chemotherapeutic agent may be one in which two or more agents are administered in combination. For example, fluoropyrimidine and deoxynucleoside analogs can be administered in combination. In another embodiment, gemcitabine and 5-fluorouracil (or capecitabine, a prodrug thereof) may be administered in combination. In another embodiment, gemcitabine and capecitabine may be administered in combination.

Appropriate dosages of the anticancer agents mentioned herein are already well known in the art and can be administered by 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 dosage may be, for example, specifically 1 μg / kg / day to 10 g / kg / day, more specifically 10 μg / kg / day to 100 mg / kg / day, and more specifically, 50 ㎍ / kg / day to 10 mg / kg / day, but is not limited to this, depending on a variety of factors, such as age, health conditions, complications of the subject to be administered Can be.

For example, gemcitabine may be administered intravenously, and the dosage and administration interval may be 1 to 6 times weekly at 2 to 8 week intervals at a dose of 100 to 10,000 mg / m ² . Dosage is at least 100 mg / m ² , at least 200 mg / m ^{2, at} least 300 mg / m ^{2, at} least 400 mg / m ^{2, at} least 500 mg / m ^{2, at} least 600 mg / m ² , at 700 mg / m ² Or more, 800 mg / m ² or more, or 900 mg / m ^{2 or} more. Or, the dosage is 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, 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 Or less, 1050 mg / m ² or less, 1030 mg / m ² or less, 1020 mg / m ² or less, or 1010 mg / m ² or less.

In the case of capecitabine, it may be administered orally. The dosing interval may be twice daily. Doses are at least 500 mg / m ^{2, at} least 600 mg / m ^{2, at} least 700 mg / m ^{2, at} least 800 mg / m ² , 900 mg / m ² , 1,000 mg / m ² , 1,100 mg / m 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. Or, the dosage is 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 It may be: The daily dose may be administered two or more times divided.

In one aspect, the peptides and / or anticancer agents described herein can 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 what is added to the vaccine to stimulate an immune response to the target antigen, but does not itself provide immunogenicity. All vaccines cause inflammation, resulting in the accumulation of bone marrow cells (monocytes and Neutrophils). Some of these cells are MDSCs. In addition to the purpose of stimulating the immune response, there are also adjuvants added to stabilize the formulation of the vaccine. Immunological adjuvant is well known in the art [J Biomed Biotechnol. 2012; 2012: 831486. Published online Mar 13, 2012]. Immunological adjuvant includes inorganic adjuvant such as aluminum salt and organic adjuvant such as oil based, virosome, squalane. Organic adjuvants include, but are not limited to, emulsions, microbe-derived, synthetic adjuvants, cytokines, and the like. 9 kinds of cytokine adjuvant are known. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF), which activates mature granulosite 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 an adjuvant that increases MDSC inhibits the immune response and thus cannot achieve the effect of the vaccine. In this situation, a means is needed to prevent the increased action of MDSC of the adjuvant used.

Appropriate dosages of the adjuvant referred to 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. Specific dosage determinations are within the level of those skilled in the art, and their daily dosage may be, for example, specifically 1 μg / kg / day to 10 g / kg / day, more specifically 10 μg / kg / day to 100 mg / kg / day, and more specifically, 50 ㎍ / kg / day to 10 mg / kg / day, but is not limited to this, depending on a variety of factors, such as age, health conditions, complications of the subject to be administered Can be.

For example, in the case of GM-CSF, intradermal doses of 7 to 700 mg prior to administration of the peptides disclosed herein, such as from 1 minute to 150 minutes before, 5 to 80 minutes before, or 10 to 15 minutes before May be administered. The administration time may be administered at least 1 minute before, at least 3 minutes, at least 5 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes or at least 10 minutes before the peptide administration. In addition, 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, and before 40 minutes Before, 30 minutes or less, 20 minutes or less, or 15 minutes or less. The dosage can be at least 7 mg, at least 10 mg, at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg or at least 70 mg. In addition, the dosage may be 700 mg or less, 600 mg or less, 500 mg or less, 400 mg or less, 300 mg or less, 200 mg or less, 100 mg or less, 90 mg or less, or 80 mg or less.

GM-CSF, which is added as an adjuvant for vaccines in preclinical trials, 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, low doses of GM-CSF as a vaccine adjuvant are known to increase 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 of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 25: 2546-2553. The adjuvant added to aid the vaccine's immune function would rather interfere with the immune response by increasing the number of MDSCs. Therefore, GM-CSF has a big limitation that it is difficult to use as an adjuvant for cancer vaccine.

The present inventors have found a breakthrough method that can solve the problem of the loss of MDSC reduction ability by the combination of gemcitabine and 5-fluorouracil (or capecitabine) and the action of increasing MDSC in the tumor micro-environment of GM-CSF. Came out. We found that administration of low dose GM-CSF as an adjuvant of PEP1 did not increase Lin-DR-CD11b + MDSC in a group of patients receiving GemCap and PEP1 at the same time.

High MDSC% before vaccination does not render it impossible to elicit an immune response to tumor associated antigens.

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

Since the dosage, administration method, administration cycle, and the like of each active ingredient are already well known in the art, it may be administered according to criteria known in the art according to the condition of each patient. Specific dosage determinations are within the level of those skilled in the art, and their daily dosage may be, for example, specifically 1 μg / kg / day to 10 g / kg / day, more specifically 10 μg / kg / day to 100 mg / kg / day, and more specifically, 50 ㎍ / kg / day to 10 mg / kg / day, but is not limited to this, depending on a variety of factors, such as age, health conditions, complications of the subject to be administered Can be.

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 may be administered orally, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intradural or subcutaneous.

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, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

Pharmaceutical compositions according to one aspect of the invention may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring or sweetening agents as needed. Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods 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 term without the number before the noun is not intended to limit the quantity but rather to the presence of one or more of the mentioned noun articles. The terms "comprising", "having", and "comprising" are interpreted as open terms (ie, meaning "including but not limited to").

Reference to a range of numbers is simply an easy way to substitute for referring to each individual number within the range individually, and unless stated otherwise, each individual number is referred to individually as if It is incorporated herein as is. The end values of all ranges fall within that range and can be combined independently.

All methods mentioned herein may be performed in the proper order unless otherwise specified or clearly contradicted by context. The use of one embodiment and all embodiments or example language (eg, “such as”) is merely intended to better describe the invention, unless it is within the scope of the claims, and covers the scope of the invention. It is not intended to be limiting. No language in the specification should be construed as essential to the practice of the invention as to any unclaimed elements. Unless defined otherwise, 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 invention include the most optimal mode known to the inventors for carrying out the 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 make appropriate use of such variations, and the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any combination of the abovementioned elements within all possible variations is included in the invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims .

Example 1 Synthesis of Peptides

A peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof, was prepared according to a conventional solid phase peptide synthesis method. Specifically, peptides were synthesized by coupling amino acids one by one from the C-terminus through Fmoc solid phase synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, the first amino acid at the C-terminus of the peptides was attached to the resin. 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 were protected by Fmoc with N-term and all residues removed from acid with Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl) and the like were used. For example:

Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc- Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, 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.

Coupling reagent is HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] It was. Fmoc removal was performed using piperidine in DMF in 20% of DMF. The cleavage cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5] was used to separate the synthesized peptide from Resin and remove the protecting group of the residue. It was.

Each peptide was synthesized by repeating a process of reacting the amino acids with each other, washing with a solvent, and then deprotecting the amino acid using the state in which the amino acid protecting group was bound to the solid support. The synthesized peptide was separated from the resin and then purified by HPLC, and confirmed by MS and lyophilized.

Referring to Pep 1 (EARPALLTSRLRFIPK) consisting of SEQ ID NO: 1, the specific process is as follows.

1) coupling

Amino acid (8 equivalents) protected by NH ₂ -Lys (Boc) -2-chloro-Trityl Resin and coupling reagent HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added. Reaction was performed at room temperature for 2 hours, and washed with DMF, MeOH, and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF at 20% was added thereto, reacted twice at room temperature for 5 minutes, and washed in the order of DMF, MeOH, and DMF.

3) Peptide base skeleton NH ₂ -E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin).

4) Cleavage: A cleavage cocktail was added to the synthesized peptide Resin to separate the peptide from Resin.

5) Cooling diethyl ether was added to the mixture, followed by centrifugation to precipitate the peptide.

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

Comparative Examples 1, 2 and Example 2: Concomitant Administration

20-30 ml of venous blood were collected from 40 pancreatic cancer patients (Comparative Example 2, Example 2) and 24 healthy controls (Comparative Example 1) of age and gender matched with the patients. Controls were recruited from a surgical minor operation clinic at Royal Surrey Country Hospital in the United Kingdom. All 40 patients have never had autoimmune diseases or recent steroid treatments, and controls have never had cancer. All subjects who participated in the study submitted written informed consent and were approved by the local human investigators committee. All patients received gemcitabine and capecitabine in combination (GemCap). Forty patients were divided into two groups, arm2 (Comparative Example 2) and arm3 (Example 2). Arm2 (Comparative Example 2) was 19, and arm3 (Example 2) was 21. Arm2 (Comparative Example 2) was treated with gemcitabine and capecitabine in combination, and arm3 (Example 2) was simultaneously administered pep1 peptide and its adjuvant GM-CSF in addition to gemcitabine and capecitabine.

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

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

Example 2 (arm3): 21 pancreatic cancer patients, gemcitabine-capecitabine (GemCap) + pep1-GM-CSF administration

Gemcitabine (1,000 mg / m ² ) was administered intravenously three times weekly at four week intervals, while capecitabine was administered orally twice a day for three weeks and not for one week. The dosage 1,660mg / m ² / day was (830mg / m ² 1 twice daily). The MDSCs trajectories of 19 patients with arm2 during and after GemCap treatment and two cycles of GemCap treatment were analyzed. We also analyzed the MDSCs trajectories of 21 patients in arm3 who received both GemCap and pep1. The first pep1 was administered intra-dermally 0.56 mg of PEP1 on the 1st, 3rd, and 5th days of week 1, and the 2nd, 3rd, 4th, 6th, and 10th weeks, and 75mg GM 10-15 minutes before pep1 administration. -CSF was administered intradermally at the same site as pep1 with the adjuvant of pep1.

Peripheral blood samples were taken before and after GemCap administration. Arm2 patients receiving only gemcitabine and capecitabine were taken with blood samples after 7 weeks of treatment, before the sixth gemcitabine, and when capecitabine was administered. After 10 weeks of treatment, the 7th gemcitabine immediately and before the 8th gemcitabine, and before the capecitabine, will be consistent with the immunomonitoring timing of the arm3 patient group receiving gemcitabine, capecitabine, and pep1. When a blood sample was taken.

Three patients from Arm 3 group were taken at week 14 and two patients were taken at week 18, which coincided with the timing of pep1 administration. The collected blood samples were transferred to li-heparin tubes (BD Biosciencs, Europe) or CPT tubes to a Biomarker repository located in Liverpool Cancer Trial Unit (UK). PBMCs were separated using a Ficoll-Hypaque gradient, the cells were counted, frozen at minus 80 ° C for later analysis and stored in liquid nitrogen.

Immunophenotypic analysis of cells

Peripheral blood mononuclear blood cells were recovered using 0.15 M phosphate-buffed saline solution (Dulbecco's A) (Oxoid, UK). Subsamples of recovered cells were used for MDSC analysis and the LIVE / DEAD Cell Stain kit (Invitrogen, UK) was used to distinguish between living and dead cells. 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) Anti-human monoclonal antibodies were used for analysis using flow cytometry. Cells were immunostained, washed with 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 intradermal opposite the belly vaccine site. Patients were asked to measure and report the size of the DTH response 48 hours after intradermal injection. DTH reaction was accompanied by erythema and sclerosis, and the average diameter was 5mm.

In vitro proliferation assays

Thawed PBMCs were aliquoted into 48-well plates (ThermoFisher Scientific, USA) to be 2x10 ⁶ cells per well and then X-VIVO (Lonza) containing 10% pooled human serum (Innovative Research, USA), 20 ug / ml pep1 peptide. , UK) cultured for 3 days. After incubation, 10 units / ml IL-2 (Peprotech, UK) was added to the medium. On day 11 of cultivation, after harvesting PEP1 cells, 1 × 10 ⁵ cells per well were dispensed into round-bottom 96 well plates. 50 μl of 1 × 10 ^{5 per} well was added to pre-stimulated cells to serve as antigen-presenting cells, with pep1 specific cell proliferation to 100 μl control medium (pep1). (20 μg / ml) or positive control (5 μg / ml) was added and analyzed after two days of culture After incubation, 1 μCi of ³ H-thymidine per well was counted for 16 hours before counting the cells. A higher stimulation index (SI) of 1.8 than the number of cells per minute was determined as a positive proliferative response to pep1.

Cytokine Analysis

Cytokine levels of patient serum collected during PBMC collection were analyzed using BioRad BioPlex 27 Assay of BioRad BioPlex Instrument.

Tumor Burden assessment

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

Statistical Analysis

Welch's correction and unpaired t-test were used to compare median values of Lin-DR-CD11b + cells in advanced pancreatic cancer patients and controls. Spearman's rank test was used to analyze the association between the MDSC baseline and cytokine baseline values, and the nonparametric Mann-Withney test was used to analyze the differences in cytokine dichotomized from the median MDSC values. Paired Wilcoxon tests were used to compare cytokine levels post-chemotherapy post-treatment.

 In order to calculate the absolute value of the MDSC difference, the pre-treated MDSCs were subtracted from the post-treatment MDSC values. The calculated data are asymmetrically distributed and graphically logged at log scale, but all analyzes represent the original dimensions using a nonparametric approach. Wilcoxon signed rantks test was used to analyze differences in each treatment group. The Wilcoxon two-sample tests were used to compare pre-treatment MDSC values, post-treatment MDSC values, MDSC difference absolute values, and MDSC difference% absolute values in arm 2 and arm 3 treatment groups. Wilcoxon two-sample tests were used to compare the% absolute value of the (PD) MDSC difference between patients with disease control (PR, SD) and patients with progressive disease. For consistency of trends, sensitivity analyzes include reanalyzed data on patients who are suppressing the disease to eliminate the effects of tumor size changes, and on reconstructed data on arm3 after 10 weeks of follow-up.

result

1. Association of MDSC with Proinflammatory Cytokine Levels

As shown in FIG. 1, the reference value of Lin-DR-CD11b + cells and the level of pro-inflammatory cytokine in pancreatic cancer patients were not related.

Cryopreserved peripheral blood mononuclear cells (PBMCS) of 40 patients with advanced pancreatic cancer who received gemcitabine and capecitabine were analyzed. Twenty-one of the 40 patients received the telomerase vaccine PEP1, gemcitabine and capecitabine at the same time. The pretreatment baseline of 21 patients was used to calculate the association between pretreatment median and inflammatory cytokine MDSC levels. To analyze the effects of gemcitabine and capecitabine on Lin-DR-CD11b + cells, a sample of 19 patients who received only gemcitabine and capecitabine consecutively was analyzed. The phenotype obtained from the analysis was used to represent MDSCs based on Kotsakis' findings.

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

Inflammatory cytokines in 33 pancreatic cancer patients with data on total cytokines (Speraman's coefficient: IL-6 = 0.153, IL-1 '= 0.22, VEGF = -0.0389, TNFа = 0.0587, MCP-1 = -0.226) No association between Cain and MDSCs reference values was found. When the MDSC levels were divided into median values, no significant difference in the reference value of the cytokine was found between those with low MDSCs and those with high levels (FIG. 1).

2. Results of Comparative Example 2 (arm2) administered with GemCap only

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

Tumor volume Eight out of 19 patients (comparative example 2, arm 2) who received a combination of gemcitabine and capecitabine had decreased Lin-DR-CD11b + cell numbers. Of seven patients with advanced cancer (progressive disease, PD), five Lin-DR-CD11b + cell levels were elevated and two levels were reduced (range -60 to +662%). Of 10 patients with no advanced cancer (Stable disease, SD), 6 Lin-DR-CD11b + cell levels were elevated and 4 levels were decreased (range -68- + 604%). Patients who responded partially to the Pep1 vaccine had decreased Lin-DR-CD11b +%. The tumor size was accurately measured and no significant increase or decrease was found in the sum of the longest diameters of the significant tumors in 8 patients out of 10. The direct effect of the change in tumor volume on the 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 + was not decreased by gemcitabine and capecitabine itself. Changes in Lin-DR-CD11b +% tended to track tumor response. This has been demonstrated in patients with Lin-DR-CD11b + reference values greater than the median, and the reduction of MDSCs will be of great help immunologically in this patient group (Table 3).

TABLE 3

Number	Stage	Baseline Lin-DR-CD11b +	Post therapy Lin-DR-CD11b +	Change (%)	Radiological response
One	III	2.58	18.16	+604	SD (0%)
2	IV	3.65	4.12	+13	PD
3	III	2.25	3.33	+48	SD (0%)
4	IV	2.54	1.59	-37	SD (-11%)
5	IV	2.0	1.08	-46	SD (0%)
6	IV	2.69	3.64	+35	PD
7	IV	2.23	4.12	+85	PD
8	IV	2.96	0.94	-68	PR
9	III	1.86	2.02	+9	SD (0%)

Of the patients with a baseline value of Lin-DR-CD11b +% greater than the median, 6 had increased Lin-DR-CD11b +% and 3 had no change in tumor volume (0%). One of three patients with decreased Lin-DR-CD11b + had a partial response and a 11% tumor volume change. Among them, only Lin-DR-CD11b +% was lower than the median value after the combination of gemcitabine and capecitabine. When arm2 and arm3 were combined, the absolute value of MDSC change was related to the response to anticancer drugs ( p = 0.02), and the MDSC levels of nine patients with advanced cancer increased (median = 0.47). The MDSC level (median = -0.49) of 31 patients who had decreased.

 Different inflammatory cytokines and IL-6 during treatment were used to analyze the association between the extent of cancer-related inflammation and MDSC levels. The results are shown in Table 4.

Table 4

It was hypothesized that cancer-related inflammatory stimuli that induce sustained MDSC production were induced in a group of patients with little change in tumor volume but with increased MDSC%. During the administration of gemcitabine and capecitabine, IL-6 levels in seven of 19 patients with Arm2 increased, and four of them had advanced cancer. MDSC% in three patients with no cancer progressed. Table 4 shows the change in MDSC% for inflammatory cytokines during administration of gemcitabine and capecitabine in 10 of 19 patients with no cancer progression. IL-5 levels in four patients with reduced MDSC% decreased in all four patients, of whom one patient (Patient 8) had reduced IL-6 levels from 152.72 to 8.66 pg / ml during 7 weeks of chemotherapy. This was associated with MDSC%, which decreased from 2.54 to 1.59. The baseline MDSC levels of three patients in six non-advanced cancers with increased MDSC% were lower than the median and lower than the median after gemcitabine and capecitabine. Two patients whose MDSC baseline values were higher than the median had increased MDSC levels continuously during treatment and showed significant increases at IL-6 levels (Patients 1 and 3). Vascular Endothelial Growth Factor of Patient 3 also increased from 37.59 to 70.69 pg / ml, the only increase in VEGF levels among patients without cancer.

3. Comparison of MDSC Number Changes of Comparative Example 2 (arm2) and Example 2 (arm3)

Comparative Example 2 (arm2) administered gemcitabine-capecitabine (GemCap) to 19 pancreatic cancer patients, and gemcitabine-capecitabine (GemCap) + pep1-GM-CSF administered to 21 pancreatic cancer patients The result of comparing the change of MDSC number according to Example 2 (arm3) is shown in FIG.

Table 5

MDSC changes were analyzed and compared in the group of patients receiving only gemcitabine and capecitabine (GemCap) (Comparative Example 2, arm2) and the group of patients receiving GemCap and PEP1 (Example 2, arm3). Summary statistics comparing the MDSCs after administration of Arm2 and arm3 are shown in Table 5, and the MDSC change values are shown by logging in FIG. The pre-treatment MDSC values were higher in arm3 (Example 2) than in Arm 2 (Comparative Example 2) (p = 0.08), and in the arm 3 (Example 2) patient group, MDSC levels (( p = 0.007 and p = 0.006). for absolute and percentage change, but no significant reduction in arm2 (Comparative Example 2) patient group ( p = 0.60 and p = 0.62 for absolute and percentage change). There was little difference in MDSC values (P> 0.99) in the arm 3 patient group, and 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. There was a different trend between arm 2 and arm 3 groups, but remained consistent after 10 weeks of treatment with the SD subgroup.Comparing the number of patients with decreased MDSC%, a total of 21 patients for arm3 (Example 2) were compared. Of 19 patients showed a decrease, whereas in arm 2 (Comparative Example 2) only 8 of the 19 patients Showed a decrease.

The positive proliferation assay (the development of a positive DTH to PEP1) was analyzed in 21 arm3 patients who received GemCap and PEP1 at the same time. Nine of the 21 patients had an immune response, and eight of the nine had decreased MDSC% during treatment. The baseline value of Lin-DR-CD11b +% in 6 of 9 patients was higher than the median of patients and MDSC levels decreased.

All groups of patients with immune responses controlled radiation sickness during blood collection for analysis of proliferative responses in the same way as for MDSC analysis.

Experimental Example for the Single Dose Effect of Pep1

Both granular and mononuclear MDSCs were extracted from fresh mesothelioma tissue and subjected to inhibition assay in the microenvironment of mesothelioma. The experiment also uses Pemetrexed, which, when treated with mesothelioma, induces an senescence-associated secretory phenotype that results in STAT3 activation in tumor cells. Through this experiment, we could find out the effect of pep1 on cytokine release from tumor cells, pep1 on myeloid cell differentiation and pep1 on the inhibitory activity of MDSC.

1. Comparative Example 3-4 and Example 3-4: Effect of single pep1 administration on dendritic cell (DC) maturation

DC maturation experiments were performed using adherent monocytes obtained from PBMCs from healthy donors. 5-day culture was performed. The conditions were as follows.

Comparative Example 3: MoDCs + GM-CSF / IL4

Example 3: MoDCs + GM-CSF / IL4 + pep1

Comparative Example 4: MoDCs + GM-CSF / IL4 + pemetrexed

Example 4: MoDCs + GM-CSF / IL4 + pep1 / pemetrexed

MoDCs: Monocyte-derived Dendritic Cells

All samples were treated with or without LPS (Lipopolysaccharides) overnight. The cultured samples were tested for:

DC maturation markers;

T cell stimulating function of DC in vitro; And

Ability to inhibit T cell proliferation in CD3 / CD28.

Arginase, Inos, Ros, Stat 3 and Stat 6 were also analyzed.

2. Comparative Examples 5-6 and 5-6: Effect of DC Maturation and pep1 on Presence of Tumor-derived Factors

Mesothelioma cells were plated in 25 flasks and treated with the following samples at 40-50% confluence.

Comparative Example 5: Mesothelioma cell line (MCL)

Example 5: Mesothelioma cell line + GV1001 (MCLG)

Comparative Example 6: Mesothelioma cell line + pemetrexed (MCLP)

Example 6: Mesothelioma cell line + GV1001 / pemetrexed (MCLGP)

The treatment was performed for 24 hours, after which the medium was removed and replaced with fresh medium. After 36 hours the supernatant was collected and stored. Thereafter, the culture was incubated for 5 days using a comparative example without pep1 and an example with pep1 as a medium.

Control: MoDCs + GMCSF / IL4

Comparative Example 7: MoDCs + GMCSF / IL4 + MLC

Example 7: MoDCs + GMCSF / IL4 + MCLG

Comparative Example 8: MoDCs + GMCSF / IL4 + MLCP

Example 8 MoDCs + GMCSF / IL4 + MLCGP

All samples were treated with or without LPS overnight. The cultured samples were tested for:

DC maturation markers;

T cell stimulating function of DC in vitro; And

Ability to inhibit T cell proliferation in CD3 / CD28.

Arginase, Inos, Ros, Stat 3 and Stat 6 were also analyzed.

3. Effect of pep1 on Inhibitory Activity of MDSC

The purified MDSC from the patient was treated with pep1 (also + pemetrexed) overnight, and the cells were washed and used for the mixed lymphocyte reaction (MLR) for inhibition assay. MDSCs were plated in 96 wells and treated at each concentration for MLR the next day (CD3 / CD28 stimulation). Apart from that, the treatment was performed during MLR and the treatment was also added to T cell treatment as a control.

Claims (31)

1. As a composition for inhibiting myeloid-derived suppressor cells ("MDSC"),

   The composition comprises a peptide comprising SEQ ID NO: 1 as an active ingredient for MDSC inhibition, a peptide having 80% or more sequence homology with the peptide sequence, or a fragment thereof.

   The peptide is contained in an amount effective to inhibit the MDSC, composition for inhibiting MDSC.
2. The composition of claim 1, wherein the composition is for administration to a subject in need of inhibiting MDSC.
3. The composition of claim 1, wherein the bone marrow-derived suppressor cells are bone marrow-derived suppressor cells of a subject having a tumor.
4. According to 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, MDSC inhibition composition.
5. The composition of claim 1, wherein the composition is administered in combination with an anticancer agent.
6. The method of claim 5, wherein the anticancer agent is a chemotherapeutic agent,

   Wherein said chemotherapeutic agent is any one or more of deoxynucleoside analogs and fluoropyrimidines.
7. The method of claim 6,

   The deoxynucleoside analog is gemcitabine, and the fluoropyrimidine is 5-fluorouracil or capecitabine.
8. The composition of claim 5, wherein the composition is administered in combination with an adjuvant.
9. The composition of claim 8, wherein the adjuvant is a cytokine adjuvant.
10. 10. The composition of 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 has a phenotype of Lin-DR-CD11b +.
12. Composition for inhibiting MDSC according to any one of claims 1 to 10; And

    Kit for inhibiting MDSC containing instructions.
13. The method of claim 12, wherein the kit,

    Kit for inhibition of MDSC further comprising an anticancer agent.
14. The method of claim 13, wherein the anticancer agent is a chemotherapeutic agent,

    Wherein said chemotherapeutic agent is any one or more of deoxynucleoside analogs and fluoropyrimidines.
15. The method of claim 14,

    The deoxynucleoside analog is gemcitabine, and the fluoropyrimidine is 5-fluorouracil or capecitabine.
16. The kit of claim 12, wherein the kit further comprises an adjuvant.
17. 17. The kit of claim 16, wherein the adjuvant is a cytokine adjuvant.
18. 18. The kit of claim 17, wherein the adjuvant is granulocyte-macrophage colony-stimulating factor (GM-CSF).
19. The kit for inhibiting MDSC of claim 12, wherein the instruction manual comprises a combination of the composition for inhibiting MDSC and the anticancer agent and / or the adjuvant.
20. The kit for inhibiting MDSC of claim 12, wherein the kit for inhibiting MDSC improves, prevents or treats a disease or symptom caused by MDSC through MDSC inhibition.
21. The kit for inhibiting MDSC of claim 12, wherein the kit for inhibiting MDSC improves, prevents or treats cancer through MDSC inhibition.
22. The method of claim 21, wherein the 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.
23. As a method of 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, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof, that is effective for MDSC inhibition. Way.
24. A peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof;

    Anticancer agents; And

    MDSC inhibition composition comprising an adjuvant.
25. A peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof;

    Anticancer agents; And

    An anticancer composition comprising an adjuvant.
26. A peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof;

    Anticancer agents; And

    Cancer vaccine composition comprising an adjuvant.
27. The method according to any one of claims 24 to 26,

    The anticancer agent is a chemotherapeutic agent, the chemotherapeutic agent is any one or more of deoxynucleoside analogs and fluoropyrimidines,

    Wherein said adjuvant is a cytokine adjuvant.
28. The method of claim 27,

    The peptide is a peptide consisting of SEQ ID NO: 1,

    The deoxynucleoside analog is gemcitabine, the fluoropyrimidine is 5-fluorouracil or capecitabine,

    The adjuvant is GM-CSF.
29. A composition comprising a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof;

    Anticancer agents; And

    Anticancer kit including instructions.
30. As a method of improving, treating or preventing cancer,

    An effective amount of a peptide comprising SEQ ID NO: 1, a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof, in combination with an anticancer agent and / or adjuvant to improve, treat or A method for improving, treating, or preventing cancer, comprising administering to a subject in need thereof.
31. A peptide comprising SEQ ID NO: 1 for preparing a composition for inhibiting myeloid-derived suppressor cells ("MDSC"), a peptide having at least 80% sequence homology with the peptide sequence, or a fragment thereof Use of Phosphorus Peptides.

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