TWI658832B - Composition for inhibiting myeloid-derived suppressor cells - Google Patents

Abstract

The present invention relates to a novel use of a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having sequence homology with 80% or more of the amino acid sequence, or a peptide of the above-described fragment. More particularly, the invention relates to the use of a peptide that inhibits bone marrow-derived suppressor cells (MDSC). By inhibiting MDSC, MDSC can suppress the immune response. Effective inhibition of MDSC by using a peptide in combination with an anticancer drug and an adjuvant makes it possible to prevent, alleviate or treat diseases and symptoms caused by MDSC. In addition, a vaccine (particularly a cancer vaccine), an anti-cancer kit or an anti-cancer method can be provided without MDSC-related side effects.

Description

a composition for inhibiting bone marrow-derived suppressor cells

The present invention relates to a composition, a kit and a method for inhibiting bone marrow-derived suppressor cells containing a telomerase-derived peptide, and a telomerase-derived peptide effective for inhibiting bone marrow-derived suppressor cells to fight cancer Composition, method and use.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous families of immature bone marrow cells that are blocked by various tumor secreting factors during differentiation. MDSC inhibits the activity of cytotoxic T lymphocytes in a variety of ways.

MDSC plays an important role in protecting the host's body from the adverse effects of extreme immune responses resulting from acute and chronic infections and in inhibiting the autoimmune response of antigens released by wounds. Likewise, MDSC function for inhibiting autoimmune responses can help patients with autoimmune diseases, but MDSC proliferation involves pathological pathways. Activation is regulated by various transcription factors, thereby activating the upregulation and expression of immunosuppressive agents such as ARG1 and NOS2, and increasing the production of NO, ROS, RNS and cytokines. In particular, the accumulation of MDSC Maintaining the state of immunosuppression, thus allowing the body to be continuously exposed to allergens and/or viral infections, and causing chronic inflammation. When the body is unable to adapt to the immune response, tissue damage caused by chronic inflammation can occur.

In the preclinical trial model, as a chemotherapeutic drug that has been demonstrated to directly reduce the number of MDSCs, only two drugs exist: gemcitabine and 5-fluorouracil (5-FU). Gemcitabine has been reported to reduce the significant number of MDSCs in tumor-induced mouse spleen [Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM (2005) Gemcitabine selective eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor- Bearing animals and enhances anti-tumor immune activity. Clin Cancer Res 11:6713-6721]. It has also been demonstrated that 5-FU can substantially reduce the number of MDSCs and that the MDFU reduction of 5-FU is more severe than that of gemcitabine.

In a method of inhibiting MDSC, miR-142 and/or miR-223 ribonucleotides are administered to MDSC (WO 2013/082591). The miR-142 and/or miR-223 ribonucleotides allow MDSC to differentiate into macrophages or dendritic cells, resulting in a reduced number of MDSCs.

In another method of inhibiting MDSC, the use of bisphosphonates or CCR2 inhibitors as adjuvants has been demonstrated (WO 2011/116299). Bisphosphonates include clodronate, zoledronate, pamidronate, etidronate, and other bisphosphonate drugs. Examples of CCR2 inhibitors are RS 1028595, PF-04178903 and the like.

At the same time, in preclinical trials, granulocyte-macrophage has been demonstrated The granulocyte-macrophage colony-stimulating factor (GM-CSF) is used as a vaccine adjuvant to increase MDSC in the 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]. Also, in clinical trials, low doses of GM-CSF have been demonstrated as vaccine adjuvants 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] .

[Previous Technical Literature]

[Patent Literature]

(1) WO 2013/082591

(2) WO 2011/116299

[Non-patent literature]

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

(2) Curran MA, Allison JP (2009) Tumor vaccines expressing flt3 ligand synergies 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 invention is to inhibit MDSC.

In another aspect, the invention is directed to activating an immune response.

In another aspect, the invention is directed to activating an immune response inhibited by MDSC.

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

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

In another aspect, the invention is directed to preventing, ameliorating or treating a disease or condition associated with MDSC.

In another aspect, it is an object of the present invention to provide an anti-cancer composition, an anti-cancer kit or an anti-cancer method that does not reduce the immune response associated with MDSC.

In another aspect, it is an object of the present invention to provide a cancer vaccine that does not reduce the immune response associated with MDSC.

In an exemplary embodiment of the present invention, the present invention relates to a composition for inhibiting bone marrow-derived suppressor cells (MDSC), which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence A peptide of 80% or more sequence homology or a peptide of the above fragment, which inhibits MDSC with an effective amount of a peptide.

In an exemplary embodiment of the present invention, the present invention relates to a composition for inhibiting MDSC comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug; and an adjuvant.

In an exemplary embodiment of the present invention, the present invention relates to an anticancer composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug; and an adjuvant.

In an exemplary embodiment of the present invention, the present invention relates to a composition of a cancer vaccine comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug; and an adjuvant.

In an exemplary embodiment of the invention, the invention relates to a kit for inhibiting MDSC. The kit contains a composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having sequence homology with 80% or more of the amino acid sequence, or a fragment of the above-described fragment a peptide wherein the peptide is an active ingredient, and wherein the peptide has an effective amount for inhibiting MDSC; an anticancer drug; and instructions. In another aspect, the kit further comprises an adjuvant.

In an exemplary embodiment of the invention, the invention relates to a kit for inhibiting MDSC that prevents, alleviates or treats diseases or conditions associated with MDSC by inhibiting MDSC.

In an exemplary embodiment of the invention, the invention relates to a kit for inhibiting MDSC, which prevents, alleviates or treats cancer by inhibiting MDSC.

In an exemplary embodiment of the present invention, the present invention relates to an anti-cancer kit comprising a composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amine A peptide having a sequence homology of 80% or more of a base acid sequence or a peptide which is a fragment of the above; an anticancer drug; and a specification. The kit further comprises an adjuvant composition.

In an exemplary embodiment of the invention, the invention relates to a method of inhibiting bone marrow-derived suppressor cells (MDSC), the method comprising the step of administering to a subject in need of inhibition of MDSC a sequence number comprising an effective amount for inhibiting MDSC A peptide of the amino acid sequence of 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a peptide of the above fragment. In another aspect, the method can further comprise the step of administering an anti-cancer drug in combination with the peptide. In another aspect, the method can further comprise the step of administering an adjuvant in combination with the peptide.

In an exemplary embodiment of the present invention, the present invention relates to a method for preventing, ameliorating or treating cancer, the method comprising the steps of: administering to a subject in need of prevention, alleviation or treatment of cancer an anticancer drug and/or An effective amount of an adjuvant comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having sequence homology to 80% or more of the amino acid sequence, or a fragment thereof The peptide.

In an exemplary embodiment of the invention, the invention relates to the use of a peptide for the manufacture of a composition for inhibiting bone marrow-derived suppressor cells (MDSC), the composition comprising: an amino acid sequence comprising SEQ ID NO: 1. A peptide, a peptide having a sequence homology of 80% or more with an amino acid sequence or a peptide of the above fragment.

In an exemplary embodiment of the invention, the present invention can suppress MDSC.

In an exemplary embodiment of the invention, the invention activates an immune response.

In an exemplary embodiment of the invention, the present invention addresses the problem of suppressing an immune response by MDSC.

In an exemplary embodiment of the invention, the invention prevents, alleviates or treats diseases and conditions associated with MDSC.

In an exemplary embodiment of the present invention, the present invention may provide an anti-cancer composition, an anti-cancer kit or an anti-cancer method, which has no MDSC-related side effects.

In an exemplary embodiment of the invention, the invention provides an anti-cancer vaccine that has no MDSC-related side effects.

Figure 1 is a graph showing the relationship between MDSC and levels of pro-inflammatory cytokine. The pro-inflammatory cytokine levels between high MDSC patients and low MDSC patients show that MDSC has no relationship with pro-inflammatory cytokines.

Figure 2 is a comparison of and analysis of patient groups treated with gemcitabine and capecitabine (test case 2, team 2) and patient groups treated with GemCap and pep1 (example 2, team 3 A logarithmic scale graph resulting from a change in MDSC.

The prior art documents described in the present invention are integrated with the present invention.

As used herein, the term "inhibiting MDSC" means not only reducing the number of MDSCs, but also inhibiting the activity of MDSCs. Reducing the number of MDSCs involves not only inhibiting cell proliferation, but also destroying cells and dividing cells into other cells. Moreover, in light of biology, the term includes all mechanisms known as "suppression."

As used herein, the term "in combination with" means not only the simultaneous administration of the drug, but also the combination of the various combination therapies that follow the usual course of treatment of the drug.

The inventors of the present invention have recognized a significant increase in MDSC in cancer patients. This means that MDSC can inhibit the activity of a cancer vaccine that is administered to cancer patients and thus free of cancer treatment. For the treatment of cancer by cancer vaccines, there is a need to reduce MDSC in cancer patients.

Further, the inventors of the present invention have recognized that using a chemotherapeutic drug which has been demonstrated to directly reduce the number of MDSCs, gemcitabine combined with 5-fluorouracil (5-FU) or casitabine, and each of these drugs alone Compared to the MDSC. Specifically, MDSC is increasing among many patients. This result may limit the administration of gemcitabine in combination with 5-fluorouracil (or carbitazepine).

The peptide disclosed herein significantly reduces MDSC. By reducing MDSC, peptides can address a range of pathogenic issues associated with MDSC.

The peptide disclosed herein may be a partial fragment of telomerase or the like Things. Telomeres are known as repetitive sequences of genetic material found at the end of a chromosome to prevent damage to the chromosome or to other chromosomes. The length of telomeres is shortened each time the cell divides, and after several cell divisions, the telomere length is extremely shortened to the extent that the cells stop dividing and dying. On the other hand, it is known to extend telomeres to extend the life of cells. For example, cancer cells secrete an enzyme called telomerase, which prevents telomere shortening, which leads to cancer cell proliferation.

The peptide disclosed herein may comprise a peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90% of the peptide of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. At least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence homology.

In an exemplary embodiment of the invention, the peptide comprises a peptide associated with telomerase, in particular human (Homo sapiens) telomerase.

The peptide disclosed in the present invention may comprise the peptide of SEQ ID NO: 1 or a fragment thereof in at least one amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 A peptide having a difference in a converted amino acid, at least 6 converted amino acids, or at least 7 amino acids.

In an exemplary embodiment of the invention, the peptide consists of up to 30 amino acids.

The peptide described in SEQ ID NO: 1 is the same as in Table 1 below. The "name" in Table 1 below is used to distinguish peptides. In one aspect, the peptide of SEQ ID NO: 1 is the entire peptide of human telomerase. In a different specific embodiment of the invention, the peptide having the sequence of SEQ ID NO: 1, has SEQ ID NO: A peptide of a fragment of a peptide of the sequence or a peptide having sequence homology with 80% or more of the peptide according to the present disclosure includes by selecting and synthesizing a peptide corresponding to a relevant position within the telomerase And the synthesis of "synthetic peptides." SEQ ID NO: 2 is the amino acid sequence of the entire telomerase.

In one embodiment of the invention, the alteration in the amino acid includes modification of the physical and chemical properties of the peptide. For example, an amino acid modification can be performed to improve the thermal stability of the peptide, alter the substrate specificity, and change the optimal pH.

The term "amino acid" as used herein includes not only the 22 standard amino acids naturally incorporated into the peptide, but also the D-isomer and the modified amino acid. Thus, in a particular embodiment of the invention, the peptides herein include a peptide having a D-amino acid. In another aspect, the peptide can include non-standard amino acids, such as those amino acids that have been post-translationally modified. Examples of post-translational modifications include phosphorylation, glycosylation, thiolation (including acetylation, myristylation, palmitoylation), alkylation, carboxylation, hydroxylation, saccharification, biotinylation , ubiquitination, modification of chemical properties (eg, β -removal deamidization, deamination, and structural modification) (eg, formation of disulfide bonds). Further, the change in the amino acid includes a change in the amino acid, such as an amine group, a carboxyl group or a side chain, which occurs due to a chemical reaction during the combined process of forming a peptide conjugate with the crosslinking agent.

The peptides disclosed herein may be wild-type peptides that have been identified and isolated from natural sources. In another aspect, when compared to the peptide of SEQ ID NO: 1 or a fragment thereof, the peptide disclosed herein may be an artificial variant comprising one or more amino acids substituted, deleted, and / or insert. Amino acid changes in wild-type polypeptides (not only in artificial variants) include protein folding and/or conservative substitution of amino acids that do not significantly affect activity. Examples of conservative substitutions may be in basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamic acid and Asparagus Amino acids), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids Within the group of (glycine, alanine, serine and threonine). Amino acid substitutions that do not substantially alter a particular activity are known in the art. The most frequently occurring changes 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, Asp/Gly, and the opposite changes described above. Other examples of conservative substitutions are shown in Table 2 below.

In an exemplary embodiment of the present invention, the present invention relates to a composition for inhibiting bone marrow-derived suppressor cells (MDSC), which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence A peptide of 80% or more sequence homology or a peptide of the above fragment, wherein the peptide is an active ingredient.

In an exemplary embodiment of the present invention, the present invention relates to a composition for inhibiting MDSC comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug and an adjuvant.

In an exemplary embodiment of the present invention, the present invention relates to an anticancer composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug and an adjuvant.

In an exemplary embodiment of the present invention, the present invention relates to a composition of a cancer vaccine comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amino acid sequence of 80% or more A peptide of sequence homology or a peptide which is a fragment of the above; an anticancer drug and an adjuvant.

In an exemplary embodiment of the invention, the invention relates to a kit for inhibiting MDSC. The kit contains components for MDSC, anticancer drugs, and instructions. In another aspect, the kit further comprises an adjuvant.

In an exemplary embodiment of the invention, the kit for inhibiting MDSC prevents, alleviates or treats a disease or condition associated with MDSC by inhibiting MDSC.

In an exemplary embodiment of the present invention, a set for suppressing MDSC The group prevents, alleviates or treats cancer by inhibiting MDSC.

In an exemplary embodiment of the present invention, the present invention relates to an anti-cancer kit comprising a composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1, having an amine A peptide having a sequence homology of 80% or more of a base acid sequence or a peptide which is a fragment of the above; an anticancer drug; and a specification. The kit further comprises an adjuvant composition.

In the kit, the instructions include information on the administration of the MDSC-inhibiting composition in combination with an anti-cancer drug. In particular, the instructions include information regarding the dosage, timing, and method of administering the various drugs. In addition, the instructions include inhibition of the use of MDSC, use of anti-cancer, and use of a cancer vaccine. More specifically, the instructions include all diseases or conditions that require suppression of MDSC. Also, the instructions include information on side effects and warnings related to administration.

In an exemplary embodiment of the invention, the invention relates to a method of inhibiting bone marrow-derived suppressor cells (MDSC), the method comprising the step of administering to a subject in need of inhibition of MDSC a sequence number comprising an effective amount for inhibiting MDSC A peptide of the amino acid sequence of 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a peptide of the above fragment. In another aspect, the method can further comprise the step of administering an anti-cancer drug in combination with the peptide. In another aspect, the method can further comprise the step of administering an adjuvant in combination with the peptide.

In an exemplary embodiment of the present invention, the present invention relates to a method for preventing, ameliorating or treating cancer, the method comprising the steps of: administering to a subject in need of prevention, alleviation or treatment of cancer an anticancer drug and/or An effective amount of an adjuvant comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, having A peptide having a sequence homology of 80% or more of the amino acid sequence or a peptide of the above fragment.

In an exemplary embodiment of the invention, the cancer is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, lung cancer, blood cancer, prostate A group of cancer, adenocarcinoma, and pancreatic cancer.

In an exemplary embodiment of the invention, the invention relates to the use of a peptide for the manufacture of a composition for inhibiting bone marrow-derived suppressor cells (MDSC), the composition comprising: an amino acid sequence comprising SEQ ID NO: 1. A peptide, a peptide having a sequence homology of 80% or more with an amino acid sequence or a peptide of the above fragment.

In an exemplary embodiment of the invention, bone marrow derived suppressor cells (MDSCs) may be in a subject having a tumor. Again, the composition can be administered to a subject in need of inhibition of MDSC. MDSC inhibits the immune response by inhibiting the activity of cytotoxic T lymphocytes. MDSC has an appropriate function that inhibits an acute immune response (such as an autoimmune disease), but has the adverse effect of causing or worsening the disease or interrupting proper treatment by inhibiting the desired immune response. For example, MDSCs are greatly increased in cancer or tumor patients, such that MDSCs disable cancer vaccines by reducing the effects of administering cancer vaccines. In this case, if the number of MDSCs is effectively reduced, cancer treatment will be completed easily and efficiently.

In the present invention herein, it is apparent that the disease or condition is associated with MDSC. In the present context, the diseases and conditions described herein include all common diseases and conditions associated with MDSC. For example, bone disease: osteolytic bone Biliary disease; multiple myeloma; glioblastoma; infection: bacterial or parasitic infection; acute or chronic inflammation; traumatic stress syndrome; sepsis; transplantation; intraocular autoimmune disease; inflammatory bowel disease; Cachexia; and tuberculosis (TB), but not limited to these.

In the present invention herein, the MDSC may include all MDSCs without involving their phenotypes. A variety of phenotypes are known in the art. For example, the phenotype is selected from the group consisting of CD15, IL4Ra, CD14, CD11b, HLA-DR, CD33, Lin, FSC, DR, and SSC; optionally, CD18, CD80, CD83, CD86, HLA- I; and survival/death identifiers. For example, phenotypes include MDSC1 based on the markers IL4RA+ and CD14+; MDSC2 based on the markers IL4Ra+ and CD15+; MDSC2 based on the marker IL4Ra+; based on Lin-, HLA-DR- and CD33+ and selective based on CD18+ and HLA- MDSC3 of I+; MDSC4 based on CD14+, HLA-DR (-/Io), FSCHi and SSCim; based on markers CD11b+, CD14- and CD15+ and selectivity based on FSChi, SSCim, CD80-, CD83-, CD86- and HLA- DR-MDSC5. For example, the phenotype can be Lin-DR-CD11b+.

In the compositions described herein, the concentration of the peptide can be determined according to techniques known in the art. For example, the composition according to the invention may contain from 10 mg/L to 1000 mg/L, in particular from 10 mg/L to 500 mg/L, more specifically from 30 mg/L to 200 mg/L of amino group comprising SEQ ID NO: A peptide of an acid sequence, a peptide having a sequence homology of 80% or more with an amino acid sequence, or a peptide of the above-described fragment, but when there are different effects depending on the concentration, the concentration can be modified. When the peptide is contained within the ranges mentioned above, both the safety and stability of the composition can be met and the ranges are cost-effective. Suitable.

In the present invention herein, the dose of the active ingredient in the pharmaceutical composition may vary depending on the age, sex, weight, pathology and condition of the patient, the route of administration or the diagnosis of the prescribing physician. The dosage based on such factors can be determined within the skill of those skilled in the art, and the daily dosage (for example) can be, but is not limited to, 1 μg/kg/day to 10 g/kg/day, specifically 10 μg/kg. / day to 100 mg / kg / day, more specifically 50 μg / kg / day to 10 mg / kg / day. For example, the peptide can be administered via transdermal administration. The period of administration can be every other day, once daily, and the dosing interval can be extended. For example, administration can be carried out at week 6 or week 10 after administration in each of 1 week, 2 weeks, 3 weeks, and 4 weeks. The dose administered can be from 0.1 to 3 mg per adult. The dose administered may exceed 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.45 mg or 0.5 mg. Further, the dose may be less than 3 mg, 2.5 mg, 2.0 mg, 1.5 mg, 1.0 mg, 0.9 mg, 0.8 mg, 0.7 mg or 0.6 mg.

In an exemplary embodiment, the peptide described in the present invention can be administered in combination with an anticancer drug. Anticancer drugs include, but are not limited to, chemotherapeutic drugs and biotherapeutic drugs. Chemotherapeutic drugs can include, but are not limited to, DNA alkylating agents, antimetabolites, natural materials, and hormones. Chemotherapy drugs are antimetabolites. Metabolite means a group of molecules that inhibit the synthesis of DNA and RNA. Subtypes of metabolites can include, but are not limited to, antifolate, fluoropyrimidine, deoxynucleoside analogs, and thiopurine. Fluoropyrimidines can include, but are not limited to, fluorouracil and carbipramine. Cassizabine is a prodrug of 5-fluorouracil. Deoxynucleoside analogs can include, but are not limited to, cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, Nellarabine, cladribine, clofarabine, and pentostatin. The chemotherapy drug can be administered with two or more drugs. For example, a fluoropyrimidine can be administered in combination with a deoxynucleoside analog. In another exemplary embodiment, gemcitabine can be administered in combination with 5-fluorouracil (or prodrug cethitabin). In another exemplary embodiment, gemcitabine can be administered in combination with citataxabine.

In an exemplary embodiment, the dosage of the anticancer drug described herein is determined within the skill of the art, and the daily dose (for example) may range from 1 μg/kg/day to 10 g/kg/day, more Specifically, 10 μg/kg/day to 100 mg/kg/day, more specifically 50 μg/kg/day to 10 mg/kg/day, but not limited to such numbers and may vary depending on age, state of health, complications, and the like Factors change.

For example, in gemcitabine, it can be administered intravenously every week, and the dose and interval can be 100 to 10000 mg/m ² per week and 1 to 6 times per 2 to 8 weeks and then rest 1 week. The dose may exceed 100 mg/m ² , 200 mg/m ² , 300 mg/m ² , 400 mg/m ² , 500 mg/m ² , 600 mg/m ² , 700 mg/m ² , 800 mg/m ² or 900 mg/m ² . Or the dose may be less than ^{10000mg / m 2, 9000mg / m} 2, 8000mg / m 2, 7000mg / m 2, 6000mg / m 2, 5000mg / m 2, 4000mg / m 2, 3000mg / m 2, 2000mg / m 2, 1500mg ^{/ m 2, 1400mg / m 2} , 1300mg / m 2, 1200mg / m 2, 1100mg / m 2, 1050mg / m 2, 1030mg / m 2, 1020mg / m 2 or 1010mg / m ^2.

In carbitazepine, it can be administered via oral therapy. The dosing interval can be 2 times a day. The daily dose for administration may exceed 500 mg/m ² , 600 mg/m ² , 700 mg/m ² , 800 mg/m ² , 900 mg/m ² , 1000 mg/m ² , 1200 mg/m ² , 1300 mg/m ² , 1400 mg/ m ² , 1500 mg/m ² or 1600 mg/m ² . Or the dose may be less than ^{10000mg / m 2, 9000mg / m} 2, 8000mg / m 2, 7000mg / m 2, 6000mg / m 2, 5000mg / m 2, 4000mg / m 2, 3000mg / m 2, 2000mg / m 2, 1900mg /m ² , 1800 mg/m ² or 1700 mg/m ² . The daily dose for administration can be divided into two or more times.

In an exemplary embodiment, the peptides and/or anticancers described herein can be administered in combination with an adjuvant. Adjuvants can increase MDSC. In view of immunology, adjuvants are used to stimulate the immune response of a target antigen, but do not provide immunogenicity by themselves. Each vaccine triggers inflammation, so bone marrow cells (lymphocytes and neutrophils) are called together. MDSC is the part of the cell that has been called. In addition to stimulating the immune response, there is an adjuvant for the color stability of the vaccine. Immunological adjuvants are well known in the art [J Biomed Biotechnol. 2012; 2012: 831486. published online March 13, 2012]. Immunological adjuvants include: inorganic adjuvants such as aluminum salts; oil forms; virions; and organic adjuvants such as Squalane. Organic adjuvants include, but are not limited to, emulsions, microbial derivations, synthetic adjuvants, and cytokines. For example, granulocyte-macrophage colony stimulating factor (GM-CSF), which stimulates mature granulosa cells and macrophages, is used as a vaccine against hepatitis B, HIV and cancer [J Biomed Biotechnol. 2012; 2012] :831486. Published on March 13, 2012 online]. The adjuvant described in the present invention may be an adjuvant that increases MDSC. Since the use of an adjuvant that increases MDSC reduces the immune response, the vaccine effect cannot be achieved. In this case, a means to stop the increase in MDSC is needed.

The dosage of the adjuvants described herein is within the level of those skilled in the art, and the daily dose, for example, can range from 1 μg/kg/day to 10 g/kg/day, more specifically 10 μg/kg/ Day to 100mg/kg/day, more specific 50 μg/kg/day to 10 mg/kg/day, but not limited to these numbers and may vary depending on age, state of health, complications, and various other factors.

For example, GM-CSF can be administered via transdermal administration prior to administration of the peptide described herein (eg, 1 to 150 minutes, 5 to 80 minutes, or 10 to 15 minutes), for adults 7 GM-CSF was administered intradermally to a dose of 700 mg. Further, the administration time may be more than 1 minute, 3 minutes, 5 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes before the administration of the peptide. Again, it can be administered less than 150 minutes, 130 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 15 minutes. The dose administered may exceed 7 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg or 70 mg. Further, the dose may be less than 700 mg, 600 mg, 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 90 mg or 80 mg.

In preclinical trials, GM-CSF has been demonstrated to increase MDSC as a vaccine adjuvant [Curran MA, Allison JP (2009) Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69:7747-7755 ]. Also, in clinical trials, it has been demonstrated that low doses of GM-CSF as a vaccine adjuvant 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] . Adding adjuvant will supplement The vaccine is assisted, but it will be interrupted by the increase in the number of MDSCs. Therefore, it is difficult to use GM-CSF as an adjuvant for cancer vaccines.

The inventors of the present invention have recognized that this method can solve the problem of MDSC increase caused by GM-CSF in tumor micro-conditions and the functional loss caused by administration of gemcitabine in combination with 5-fluorouracil (or cassidabine). The inventors of the present invention have recognized that when low dose GM-CSF is used as an adjuvant for pep1, Lin-DR-CD11b+MDSC of the patient group to which GemCap and pep1 are administered is not increased.

The immune response elicited by the tumor-associated antigen was not excluded due to the high percentage of MDSC prior to administration of the vaccine.

In an exemplary embodiment, the composition can be used in medicine, cosmetics, or food.

In one embodiment of the invention, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the age, sex, weight, pathology and condition of the patient, the route of administration, or the diagnosis of the prescribing physician. The dosage based on such factors can be determined within the skill of those skilled in the art, and the daily dose (for example) can range from 1 μg/kg/day to 10 g/kg/day, in particular from 10 μg/kg/day to 100 mg/ Kg/day, more specifically 50 μg/kg/day to 10 mg/kg/day, but is not limited to these numbers, and may vary depending on age, state of health, complications, and various other factors.

In one embodiment of the invention, the composition is applicable to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs, and monkeys.

In one embodiment of the invention, the pharmaceutical composition can be administered via an oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, epidural or subcutaneous route.

Orally administered forms can be, but are not limited to, lozenges, pills, soft or hard capsules, granules, powders, solutions or emulsions. Forms for parenteral administration can be, but are not limited to, injections, drip, lotions, ointments, gels, creams, suspensions, lotions, suppositories, patches or sprays.

In one embodiment of the present invention, the pharmaceutical composition may contain additives such as a diluent, an excipient, a lubricant, a binder, a disintegrant, a buffer, a dispersant, a surfactant, a colorant, etc., if necessary. Aroma or sweetener. In one embodiment of the invention, the pharmaceutical composition can be made by conventional industrial methods in the art.

The terms used herein are intended to describe the embodiments, but are not intended to limit the invention. Terms without numbers in front of them do not limit the number, but the display may have more than one thing about the terms used. The terms "including", "having", "including" and "including" (that is, "including but not limited to") should be interpreted in an open manner.

The use of a range of values is not a single number within the scope of the description, and therefore, unless explicitly stated, the range is to be construed as a End values for all ranges are included in this range and can be combined independently.

All methods discussed herein can be performed in an appropriate order unless otherwise stated or clearly contradicted in the context. The use of any embodiment and all examples or exemplary language (e.g., "such as" or "similar") is used to describe the invention more clearly, and not to limit the invention. category. Any language outside the scope of the patent application herein should not be construed as a limitation of the invention. Unless otherwise defined, the techniques used in this article The technical and scientific terms have the meanings commonly understood by those skilled in the art to which the invention pertains.

The preferred embodiment of the invention includes the best mode known to the inventors for carrying out the invention. Variations in the preferred embodiment will become apparent to those skilled in the art after reading the above description. The inventors intend for the skilled artisan to practice the invention in a variety of manners and in other ways that may vary. Accordingly, the present invention includes equivalents, modifications, and variations of the key points of the invention as set forth in the appended claims. In addition, all possible variations within any combination of the components discussed above are included in the present invention unless otherwise stated or contradicted in the context. Although the present invention has been described and illustrated by the embodiments of the present invention, it will be understood by those skilled in the art, in the form and details without departing from the spirit and scope of the invention as defined by the following claims. There can be various changes in it.

[invention mode]

Example 1: Synthesis of peptides

The peptide of SEQ ID NO: 1 was synthesized according to a conventional method of solid phase peptide synthesis. More specifically, the peptide was synthesized by coupling various amino acids from the C-terminus via Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon ROK). Their having a first amino acid at the C-terminus of the peptide attached to the resin, as follows: NH ₂ -Lys (Boc) -2- chloro group - trityl resin

NH ₂ -Ala-2-chloro-trityl resin

NH ₂ -Arg(Pbf)-2-chloro-trityl resin

Protection of all amine groups of the peptide to be synthesized by Fmoc at the N-terminus Acid, and by Trt, Boc, t-Bu (t-butylester; tert-butyl ester), Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran) soluble in acid 5-sulfonyl) protects the amino acid residue. Examples include the following: 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.

HBTU[2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate]/HOBt[N-hydroxybenzotriazole]/NMM[4 -Methylmorpholine] is used as a coupling agent. Fmoc was removed using acridine in 20% DMF. In order to remove the protection from the residue or to separate the peptide from the resin, a decomposition mixture [TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol; ethanedithiol) is used. /H ₂ O=92.5/2.5/2.5/2.5].

The peptide synthesis is carried out by using a solid phase scaffold and repeating the following process: starting from the protection of the amino acid, the individual amino acids are reacted separately, washed and deprotected with a solvent. Each peptide is synthesized by reacting the corresponding amino acid alone, using solvent washing and deprotection, and repeating the processes by using a solid phase scaffold combined with the starting amino acid and amino acid protection. After release from the resin, the synthesized peptide was purified by HPLC, confirmed by mass spectrometry, and lyophilized, and verified by MS for synthesis, and then lyophilized.

The purity of the prepared peptide was found to be 95% by high performance liquid chromatography or higher.

The specific peptide synthesis process is described based on the synthesis process of PEP 1 having SEQ ID NO: 1, as shown below.

1) Coupling an amino acid (8 equivalents) protected with NH ₂ -Lys(Boc)-2-chloro-trityl resin and a coupling agent HBTU (8 equivalents) / HOBt (8 equivalents) melted in DMF /NMM (16 equivalents) were mixed together and incubated for 2 hours at room temperature (RT). After incubation, the reaction mixture was subjected to sequential washing with DMF, MeOH and DMF.

2) Fmoc deprotection 20% Acridine in DMF was added and incubated for 5 minutes at RT, 2 times, followed by successive washings with DMF, MeOH and DMF.

3) The basic framework of the peptide is prepared by repeating the reactions 1) and 2) discussed above. NH ₂ -E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR (Pbf)-FIPK(Boc)-2-chloro-trityl resin.

4) Decomposition: The decomposition mixture is added to the fully synthetic peptide, thereby separating the synthesized peptide from the resin.

5) Pre-cooled diethyl ether was added to the obtained mixture, and then centrifuged to precipitate the collected peptide.

6) After purification by preparative HPLC, the molecular weight was confirmed by LC/MS and lyophilized to give a powder form.

Test Examples 1, 2 and 2: Combination administration

From pancreatic cancer patients (n=40) (test case 2 and case 2) and age / Gender matched normal healthy controls (n=24) collected 20-30 ml of venous blood. Age- and gender-matched healthy controls were recruited by the surgical minor surgery clinic at Royal Surrey County Hospital, UK. None of the subjects had a history of autoimmune disease or recent steroid therapy and the uncontrolled donor had a prior history of cancer. Written informed consent from the local human resources investigation committee was provided to all subjects. All patients were treated with a combination of gemcitabine and carbathionine chemotherapy (GemCap). All 40 patients were divided into two groups of Team 2 (Test Case 2) and Team Group 3 (Example 2). Group 2 (test case 2) was treated with a combination of gemcitabine and cititabine, and pep1 peptide and GM-CSF were simultaneously used as adjuvants to the combination of gemcitabine and cititabine treatment team 3 (Example 2).

- Test Example 1 (control group): 24 normal healthy controls treated with combination gemcitabine-carbitabine (GemCap)

- Test Example 2 (Team 2): 19 patients with pancreatic cancer treated with combination gemcitabine-carbitabine (GemCap)

- Example 2 (Team 3): 21 patients with pancreatic cancer treated with combination gemcitabine-carbitabine (GemCap) + pep1-GM-CSF

1000 mg/m ^{2 of} gemcitabine was administered intravenously every week, three times every 4 weeks and then one week rest, and oral administration of citridabine 3 at 1660 mg/m ² /day (830 mg/m ² each time, twice daily) Week, rest for 1 week. The inventors analyzed the MDSC trajectories of 19 patients treated in team 2 during the first two cycles of GemCap treatment. The inventors analyzed the MDSC trajectories of 21 patients who received pep1 in combination with GemCap in team 3. The main pep1 course consists of the following: on the first day, the third day, the fifth day, and then on the week of the second week, the third week, and the fourth week, followed by the sixth week and Subsequently, 0.56 mg of pep1 was administered intradermally for the 10th week. 10-15 minutes before administration of each pep1 at the same site, GM-CSF was used as an adjuvant, and a dose of 75 mg was administered intradermally.

Peripheral blood samples were taken prior to administration of GemCap and after administration of GemCap. For patients receiving gemcitabine and cecitabine alone (team 2), blood was drawn before the sixth gemcitabine infusion after 7 weeks of treatment. In order to be consistent with the time of immunization monitoring, in the patients receiving gemcitabine and carbipine with pep 1 (team 3), after the first 10 weeks of treatment, the seventh gemcitabine infusion will be performed for the eighth time after 1 week. Blood was drawn before gemcitabine infusion.

In team 3, blood was drawn from 3 patients at week 14 and blood was drawn from 2 patients at week 18, which was consistent with delivery of pep1. Blood was introduced into lithium heparin tubes (BD Biosciences, Europe) or CPT tubes for shipment to a biomarker repository of Liverpool Cancer Trials Unit, UK, counting PBMC, frozen at -80 ° C, and stored in liquid N ₂ for Subsequent batch analysis.

Immunophenotypic analysis of cells

Peripheral blood mononuclear cells were obtained and washed with Dubuques A (Oxoid, UK) 0.15 M phosphate buffered saline. Cell aliquots were used for MDSC analysis. Live cells and dead cells were distinguished using the LIVE/DEAD Cell Stain Kit (Invitrogen, UK). After washing with binding buffer (BD Biosciences, Europe), the following anti-human monoclonal antibodies were used for flow cytometry: anti-HLA-DR-APC-Cy7, anti-Lin1 (CD3, 14, 16, 19, 20, 56) )-FITC and anti-CD11b-PECy7 (BD Biosciences, Europe). After immunostaining, wash cells with binding buffer and use software with MACSQuantify Cells were analyzed by a MACSQuant flow cytometer (Miltenyi Biotec).

Delayed-type hypersensitivity (DTH) skin test

Pep1 (100 μg) was administered intradermally at the lower abdomen opposite the vaccinated site. After 48 hours of dosing, the patient is asked to record the magnitude of the DTH response and report it to the clinician. A positive DTH response was defined as erythema and induration with an average diameter of 5 mm.

In vitro proliferation assay

In X-VIVO 15 (Lonza, UK) with 10% pooled human serum (Innovative Research, USA) and 20 μg/ml pep1 peptide in 2 x 10 ⁶ cells/well in 48 well plates (Thermo Fisher Scientific, USA) The thawing PBMC is planted in the middle. After 3 days of culture, 10 units/ml IL-2 (Peprotech, UK) was added to the medium. On day 11, pep1 enriched cells were obtained and loaded into cells at 1 x 10 ⁵ cells/well (50 μl) in round bottom 96-well plates. For the pre-stimulation cells, irradiation (45 Gy) autologous PBMC (1 × 10 ⁵ cells/well, 50 μl) was added to serve as antigen-presenting cells (APCs). Pep1-specific proliferation was tested by the addition of 100 μl of control medium, pep1 (20 μg/ml) or positive control PHA (5 μg/ml). After 2 days of further incubation, 1 μCi/well of 3H-thymidine was added for 16 hours before counting. A positive proliferative response to pep1 was defined as a stimulation index (SI) of 1.8 or more and a significant difference in counts per minute among the four replicates.

Cytokine analysis

Follow the manufacturer's instructions, use BioRad BioPlex Instrument, use BioRad BioPlex 27 Pro Cytokine, Chemokine And Growth Factor Assay (BioRad Laboratories, USA) assays the cytokine levels of patient serum collected simultaneously with PBMC.

Tumor burden assessment

Radiologists blinded to MDSC results used the RECIST v1.1 standard to measure evaluable lesions on CT images to perform independent assessments before and after chemotherapy. The tumor burden was measured in millimeters using the sum of the long-axis measurements of the tumor lesions and the short-axis measurements of the pathological lymph nodes.

Statistical Analysis

The median level of Lin-DR-CD11b+ cells in pancreatic cancer patients and controls was compared using an unpaired t test with Welch correction. The association between baseline MDSC and cytokines was analyzed using the Spearman rank test, and when the scores were scored at the median MDSC level, significant differences in cytokines were identified using the non-parametric Mann-Whitney test. The cytokine levels before and after chemotherapy were compared using the paired Wilcoxon test. The value of MDSC before treatment is subtracted from the value of MDSC after treatment to give an absolute change in MDSC, and the percentage change is calculated by dividing the absolute change by the value before treatment. These data are highly skewed and therefore graphically represented on a logarithmic scale, but all analyses are performed on the original scale using the parent-free method. Changes in each treatment group were tested using the Wilcoxon marker rating test. The Wilcoxon two-sample test was used to compare Team 2 and Team 3 for pre-treatment values, post-treatment values, absolute changes, and percent changes. For the absolute and percentage changes, patients with disease control (PR, SD) and progressive disease (PD) were compared using the Wilcoxon two-sample test. Sensitivity analysis for trend continuity includes: (1) Reanalysis including only SD patients to remove tumor size changes The effect of the change, and (2) reanalysis of patients in team 3 that included only 10 weeks post-treatment time series.

result

1. Relationship between pro-inflammatory cytokines and the level of MDSC

As shown in Figure 1, in patients with pancreatic cancer, the level of pro-inflammatory cytokines is independent of the baseline level of Lin-DR-CD11b+ cells.

Frozen cryopreserved PBMC were analyzed in 40 patients with advanced pancreatic cancer treated with gemcitabine and carbitabine chemotherapy. Twenty-one of the patients received concomitant therapy with the telomerase vaccine pep1. Baseline pre-treatment values for these patients included the calculation of the median pre-treatment and the association of baseline levels of pro-inflammatory cytokines with MDSC levels. In analyzing the effects of gemcitabine and carbitalopibine on Lin-DR-CD11b+ cells, only serial samples from 19 patients receiving gemcitabine and carbitatripin alone were analyzed. This phenotype is based on Kotsakis and peer studies used to label MDSC.

The Lin-DR-CD11b+ cells in pancreatic cancer patients (n=40) were significantly increased compared to the control (test example) (p<0.0001). The median baseline of Lin-DR-CD11b+ cells in patients (expressed as % live PBMC) was 1.85 (pitch 0.62-8.45); the corresponding value in 24 controls was median 0.82 (pitch 0.16-2.2) .

There was no association between baseline levels of pro-inflammatory cytokines in 33 patients with pancreatic cancer with complete cytokines data and baseline MDSC (Spearman coefficient: IL-6 = 0.153, IL-1β = 0.22, VEGF = -0.0389, TNFα = 0.0587, MCP-1 = -0.226). When MDSC levels are halved at the median, patients with high MDSC and those with low MDSC In comparison, there was no significant difference in baseline levels of these cytokines (Figure 1).

2. Results of only test case 2 (team 2) of GemCap

Due to GemCap therapy of Test Example 2 (Team Group 2), gemcitabine and carbitabine therapy did not consistently reduce the number of Lin-DR-CD11b+ cells, the contribution to disease control and the extent of cancer-related inflammation. More specifically, it is as follows.

In patients receiving chemotherapy alone (Test Example 2, Team 2), gemcitabine and carbitabine therapy resulted in a decrease in Lin-DR-CD11b+ cells in 8 of 19 patients. Among the 7 patients with worsening conditions (PD), 5 patients had increased Lin-DR-CD11b+ cell levels and 2 patients decreased (pitch -60 to +662%). Of the 10 patients with stable disease (SD), 6 patients had increased Lin-DR-CD11b+ cell levels and 4 patients had decreased (pitch -68 to +604%). Both types of patients who achieved partial response to the pep1 vaccine decreased in percentage of Lin-DR-CD11b+. The inventors have obtained accurate tumor measurements, and in 8 out of 10 patients with stable disease, there is no significant increase or decrease in the sum of the longest diameters. The relative contribution of the percentage change in Lin-DR-CD11b+ as a direct result of any significant change in tumor size would be minimal among those patients, with a rise in the percentage of Lin-DR-CD11b+ in 5 patients and a decrease in 3 patients. These data indicate that there is no consistent reduction in the percentage of Lin-DR-CD11b+ secondary to gemcitabine and carbitabine chemotherapy itself. The percentage change in Lin-DR-CD11b+ has a tendency to track tumor response. This is a good demonstration that in patients with a baseline Lin-DR-CD11b+ greater than the median, the group with decreased MDSC would most likely have the greatest immunological benefit (Table 3).

Among these patients, the percentage of Lin-DR-CD11b+ increased in 6 patients. Three of these patients had progressive disease as the best response to therapy, and 3 had a 0% tumor volume change. Of the 3 patients with a decrease in the percentage of Lin-DR-CD11b+, 1 patient achieved partial response and another patient had a 11% reduction in tumor volume. In only 3 of these patients, the percentage of Lin-DR-CD11b+ was lower than the baseline median after gemcitabine and carbitabine therapy. When combining team 2 and team 3, the absolute change in MDSC was associated with a response (p = 0.02), with a mean increase in the level of 9 patients with PD in the study (median = 0.47) and disease control The level of 31 patients was reduced (median = -0.49).

The inventors next analyzed whether changes in MDSC levels that track changes in the extent of cancer-associated inflammation were used as surrogate changes in IL-6 and other inflammatory cytokines during treatment. The results are shown in Table 4.

The inventors hypothesized that in patients with tumor volume stability but an increased percentage of MDSC, there is persistent tumor-associated inflammation that continues to drive MDSC production. Seven of the 19 team 2 patients had elevated IL-6 levels during gemcitabine and cititabine treatment; 4 of these patients deteriorated. The other 3 patients had stable disease and the percentage of MDSC increased in these 3 patients. Table 3 shows the percent change in MDSC relative to changes in inflammatory cytokines during chemotherapy for 10 of the 19 patients with stable disease. Of the four patients with a decreased MDSC percentage, IL-6 decreased in all four patients, and in one of these patients (patient No. 8), IL-6 levels decreased from 152.72 pg/ml after 7 weeks of chemotherapy. 8.66 pg/ml was associated with a decrease in MDSC percentage from 2.54% to 1.59%. Of the six patients with stable disease with an increased percentage of MDSC, 3 patients had baseline MDSC levels below the median and remained as follows after chemotherapy. There was a significant increase in IL-6 (patients 1 and 3) in two patients with a median MDSC level above the median that continued to rise after treatment. Patient No. 3 VEGF levels also increased from 37.59 pg/ml to 70.69 pg/ml, the only patient with no worsening condition but showing this increase.

3. Comparative Test Example 2 (Group 2 Team) Example 2 (team group 3) the number of changes between the MDSC

The results shown in Fig. 2 and Table 5 are Example 2 (team group 3) of treating 19 pancreatic patients with GemCap (team 2) and treating 21 pancreatic patients with GemCap + pep1-GM-CSF.

The inventors analyzed the trajectories of MDSCs in patients who received GemCap alone (team 2) and those who received GemCap and pep1 vaccinated with low doses of GM-CSF as adjuvant (team 3), and compared the two . Table 5 shows a summary of the MDSC values for pre- and post-treatment of Team 2 and Team 3, and plots the values on a logarithmic scale in Figure 1. The pre-treatment MDSC value of team 3 was higher than team 2 (p=0.08). In team 3, MDSC decreased significantly from this higher level (absolute change and percentage change were p=0.007 and p=0.006, respectively), while there was no significant change in team 2 (absolute change and percentage change were respectively p =0.60 and p=0.62). This gives There were no significantly different values for Team 2 and Team 3 after treatment (p>0.99). In terms of MDSC changes, this difference behavior between Team 2 and Team 3 has statistically significant boundaries (absolute change and percentage change are p=0.04 and p=0.06, respectively). The sensitivity analysis of the observed trends in the difference between the display team 2 and the team 3 was consistent for the post-treatment evaluation of the SD subgroup and the 10-week subgroup.

The inventors analyzed the development of a pep1 immune response (development of a positive proliferation test for pep1 and/or development of positive DTH) in 21 patients who received a team of 3 with gemcitabine and cecitabine and pep1. Nine patients in this team developed an immune response. In 8 of these 9 patients, the percentage of MDSC decreased during chemoimmunotherapy. Six of the nine had a baseline LinDR-CD11b+ percentage greater than the median of the patient, and in all of these patients, the MDSC level decreased (Supplementary Table 1). When extracting blood for analysis of proliferative responses, all immunoreactors have radiological disease control (PR or SD) at a time consistent with the timing of successive MDSC assays in such patients.

Example of effect processing with pep1 alone

Microsurgical mesothelioma inhibition assays were performed by using granular and monocyte MDSCs collected from fresh mesothelioma tissue. When used for mesothelioma treatment, this assay also uses pemetrexed to induce a senescence-associated phenotype of STAT3 in activated tumor cells. By this, this assay has recognized the effect of pep 1 on the release of cytokines from tumor cells, differentiation of bone marrow cells, and inhibition of MDSC activation.

1. Test Example 3-4 and Example 3-4: Effect of dendritic cell (DC) maturation with pep1 alone

Performed by adhering monocytes using PBMCs from healthy donors DC maturation experiment. The culture was carried out for 5 days. The conditions are as follows.

Test Example 3: MoDCs+GM-CSF/IL4

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

Test Example 4: MoDCs+GM-CSF/IL4+ Pemetrexe

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

*MoDCs: monocyte-derived dendritic cells

All samples were treated with LPS (lipopolysaccharide; Lipopolysaccharides) for 1 night or not. The samples cultured in the test were as follows.

DC maturation marker; T cell stimulation function of DC in vitro; and inhibition ability in CD3/CD28 T cell proliferation.

Glutamate, Inos, Ros, Stat3 and Stat6 were also analyzed.

2. Test Examples 5-6 and Examples 5-6: Effect of pep1 with or without tumor-derived factors and maturation of DC

Mesothelioma cells were loaded into 25 flasks and treated with 40%-50% confluent with the following samples.

Test Example 5: Mesothelioma cell line (MCL)

Example 5: Mesothelioma cell line + pep1 (MCLG)

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

Example 6: Mesothelioma cell line + pep1/pemetrexed (MCLGP)

During the treatment, after 24 hours of treatment, the used medium was removed and replaced with new medium. After 36 hours, the culture supernatant was collected and stored. Subsequently, culture was carried out for 5 days using a test example which was not treated with pep1 and an example of treatment with pep1 as a medium.

Control: MoDCs+GMCSF/IL4

Test Example 7: MoDCs+GMCSF/IL4+MLC

Example 7: MoDCs+GMCSF/IL4+MCLG

Test Example 8: MoDCs+GMCSF/IL4+MLCP

Example 8: MoDCs+GMCSF/IL4+MLCGP

All samples were treated with LPS (lipopolysaccharide) for 1 night or left untreated. The samples cultured in the test were as follows.

DC maturation marker; T cell stimulation function of DC in vitro; and inhibition of T cell proliferation at CD3/CD28.

Glutamate, Inos, Ros, Stat3 and Stat6 were also analyzed.

3. The effect of activated pep1 on inhibiting MDSC

Purified MDSC from the patient was treated overnight with pep1 (also + pemetrexed) and used for MLR (Mixed lymphocyte reaction) after washing to suppress the assay. On the other hand, treatment and addition to T cell treatment were performed as controls at the time of MLR.

<110> Hanshang. Keljem Vickers Co., Ltd.

<120> A composition for inhibiting bone marrow-derived suppressor cells

<130> OF14P079/TW

<160> 2

<170> PatentIn version 3.2

<210> 1

<211> 16

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 1132

<212> PRT

<213> Homo sapiens

<400> 2

Claims (7)

A use of a peptide for the preparation of a composition for activating an immune response inhibited by MDSC in a subject having a tumor by inhibiting bone marrow-derived suppressor cells (MDSC), the composition Including: a peptide consisting of the amino acid sequence of SEQ ID NO: 1, the composition inhibiting MDSC with an active amount of the peptide, wherein the composition is combined with an anticancer drug and an adjuvant The administration is in combination, wherein the anticancer drug is a deoxynucleoside analog and a fluoropyrimidine, and wherein the adjuvant is a cytokine adjuvant. The use according to claim 1, wherein the tumor is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), gastric cancer (GC), melanoma, lung cancer, blood cancer, prostate cancer, adenocarcinoma, and pancreatic cancer. The group that makes up. The use of claim 1, wherein the fluoropyrimidine is 5-fluorouracil or carbitatrip. The use of claim 3, wherein the fluoropyrimidine is citamycin. The use of claim 1, wherein the deoxynucleoside analog is gemcitabine. The use of claim 1, wherein the cytokine adjuvant is a particulate cell-macrophage colony stimulating factor (GM-CSF). The use of any of claims 1 to 6, wherein the MDSC has a Lin-DR-CD11b+ phenotype.