Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the present invention relates to a composition for enhancing cancer immunity, and more particularly to a composition containing a VIPR2 antagonist peptide for enhancing cancer immunity.
• Immune checkpoint inhibitors which mainly promote the activation of T cells (e.g., CD8-positive T cells), are known as drugs for enhancing cancer immunity, but their effects are limited to solid cancers such as colorectal cancer and pancreatic cancer. It's not enough for that.
• TAE tumor microenvironment
• TAM tumor-associated macrophages
• TAMs are divided into M1-like and M2-like phenotypes, depending on their ability to secrete cytokines and chemokines.
• M1-like TAMs are tumor-suppressive macrophages that contain a large amount of class II major histocompatibility complex (MHCII) and, as antigen-presenting cells, activate the function of T cells against cancer.
• MHCII major histocompatibility complex
• M2-like TAMs then release anti-inflammatory cytokines and growth factors, which aid tumor growth.
• an increase in M2-like TAM is associated with a decrease in patient survival rate.
• a high ratio of M1/M2 macrophages in the tumor microenvironment correlates with improved prognosis of colorectal cancer patients. Therefore, therapies that repolarize M2 macrophages to the M1 phenotype are considered beneficial for tumor treatment.
• VIP Vasoactive intestinal peptide
• VIPR1 and VIPR2 also known as VPAC1 and VPAC2
• VPAC1 and VPAC2 activates the expression of anti-inflammatory cytokine genes in mouse and human macrophages after LPS culture, and conversely activates the expression of inflammatory cytokine genes. suppress.
• inhibiting VIP signals promotes the proliferation and function of CD8-positive T cells and provides a good prognosis in viral infection and lymphoma models.
• the role of VIPR1 and VIPR2 on macrophage polarization and function in tumor models is still unclear.
• Non-Patent Document 1 and Patent Document 1 KS-133 (disclosed in Non-Patent Document 2, referred to as Seq-10 (SEQ ID NO: 2) in the examples described later), which is a representative bicyclic peptide, has been shown to improve cognitive function in psychiatric disease model mice. It has also been reported that the decline is suppressed (see Non-Patent Document 2).
• the problem to be solved by the present invention is to provide a novel means for enhancing cancer immunity, for example, by investigating the role and mechanism of VIPR2 signals on the polarization and function of tumor-associated macrophages.
• the present invention was made to solve the above problems, and was created based on the discovery that VIP antagonists play a role in enhancing the function of macrophages against cancer, at least through VIPR2. That is, the present invention provides a means for promoting tumor suppressive polarization (M1 polarization) of tumor-associated macrophages by inhibiting the function of VIPR2, and specifically includes the following embodiments.
• M1 polarization tumor suppressive polarization
• Formula (1) c[X 1 -Pro 2 -X 3 -Tyr 4 -Leu 5 -Pro 6 -c (X 7 -X 8 -Leu 9 -Cys 10 ] -X 11 ) -X 12 -X 13 (1)
• X 1 represents cysteine, Mpa (3-mercaptopropionic acid), or D-cysteine
• X 3 represents N-methylated glycine, N-methylated alanine, 2-azetidine-2-carboxylic acid
• X 8 represents tyrosine, proline or arginine
• X 7 and X 11 represent lysine and aspartic acid, ornithine and glutamic acid
• asparagine Represents any combination of acid and lysine, glutamic acid and ornithine, lysine and glutamic
• X 1 represents cysteine
• X 3 represents proline or serine
• X 7 represents lysine
• X 8 represents tyrosine
• X 11 represents aspartic acid.
• X 12 and X 13 each independently represent leucine, isoleucine or norleucine, the N-terminal amino group is acetylated, and the C-terminal carboxy group is amidated.
• the composition is preferably used to promote tumor-suppressive polarization (M1 polarization) of tumor-associated macrophages, i.e. to increase the M1/M2 macrophage ratio, and is preferably used to promote tumor-suppressive polarization (M1 polarization) of tumor-associated macrophages, i.e., to increase the M1/M2 macrophage ratio, and is used to treat solid cancers, e.g. It can be used to treat cancers (such as colorectal cancer) for which treatment is being attempted.
• M1 polarization tumor-suppressive polarization
• M1 polarization tumor-suppressive polarization
• M1 polarization tumor-suppressive polarization
• solid cancers e.g. It can be used to treat cancers (such as colorectal cancer) for which treatment is being attempted.
• composition of the present invention for example, by inhibiting the function of VIPR2, it is possible to promote tumor suppressive polarization (M1 polarization) of tumor-associated macrophages, thereby providing a novel means for enhancing cancer immunity.
• M1 polarization tumor suppressive polarization
• Figure 1 shows the influence of the culture supernatant of the mouse colon cancer cell line CT26 (CT26-CM) on the polarization of mouse macrophages, RAW264.7 cells, into M2 macrophages and the expression of VIPR1 and VIPR2 (also known as VPAC1 and VPAC2).
• CT26-CM mouse colon cancer cell line
• VIPR1 and VIPR2 also known as VPAC1 and VPAC2
• A M1 macrophage marker
• B M2 macrophage marker
• C immune checkpoint marker
• D VIPR1 and VIPR2 gene expression evaluated by real-time PCR This is the result.
• E is a representative Western blot image showing protein expression levels of VIPR1 and VIPR2 in RAW264.7 cells cultured in control medium or CT26-CM for 4 days.
• FIG. 2 shows the results of evaluating the effects of antagonists acting on both VIPR1 and VIPR2 on the polarization of RAW264.7 cells cultured in CT26-CM.
• A is M1 macrophage marker
• B is M2 macrophage marker
• C is the result of relative gene expression of immune checkpoint marker.
• D Phagocytosis (% gate) of macrophages
• E Phagocytosis (% gate) of macrophages.
• Figure 3 shows the results showing that an antagonist that acts on both VIPR1 and VIPR2 suppresses the proliferation of subcutaneously transplanted mouse colon cancer cell line CT26 in immunodeficient SCID mice lacking T cells and B cells. .
• FIG. 4 shows that when an antagonist that acts on both VIPR1 and VIPR2 was administered subcutaneously alone or in combination with a mouse anti-PD-1 antibody, subcutaneously transplanted mouse colon These results show that the proliferation of cancer cell line CT26 is suppressed.
• FIG. 5-1 shows the results showing the polarization (Relative gene expression) of macrophages when the expression of VIPR1 or VIPR2 was knocked down with each siRNA (A to C).
• Figure 5-2 shows macrophage polarization (Relative gene expression: The results show relative gene expression).
• FIG. 6 shows the results of human colon cancer specimens confirming that tumor-associated macrophages exist in the tumor stroma and their degree of infiltration is higher in correlation with the expression intensity of VIPR2 than that of VIPR1.
• FIG. 8 shows the results of evaluating the effect of VIPR2-selective antagonist KS-133 on the polarization of RAW264.7 cells cultured in CT26-CM.
• A is the M1 macrophage marker
• B is the relative gene expression of the M2 macrophage marker.
• FIG. 10 is an image of tumors excised from mice in each administration group after the anticancer test of KS-133 was completed.
• a peptide refers to a compound in which two or more amino acids are bonded through an amide bond (peptide bond), and may be, for example, a compound in which 2 to 20 amino acids are bonded through an amide bond. Further, according to the convention for peptide marking, the left end is the N-terminus (amino terminal), and the right end is the C-terminus (carboxy terminus). The first carbon atom adjacent to the carbonyl group that forms the peptide bond is called the C ⁇ carbon.
• amino acid or derivative thereof is used in its broadest sense, and includes natural amino acids as well as artificial amino acids with non-natural structures and characteristics known in the art that are characteristic of amino acids. It also includes chemically synthesized compounds and carboxylic acids with functional groups.
• unnatural amino acids include D-amino acids, ⁇ / ⁇ -disubstituted amino acids whose main chain structure differs from the natural type (such as ⁇ -methylated amino acids such as 2-aminoisobutyric acid), N-alkyl-amino acids (N-methyl amino acids, etc.), N-substituted glycine (peptoids), amino acids with extended main chains ( ⁇ homoamino acids and ⁇ homoamino acids), amino acids with side chain structures different from the natural type (cyclohexylalanine, allylglycine, 2- (2-pyridyl)-glycine, 3-(1H-benzimidazol-2-yl)-alanine, etc.), amino acids with partially substituted side chains (norleucine, diaminopropanoic acid, 3-(2-pyridyl), etc.) Amino acids with extra functional groups in their side chains (such as homonorleucine and ⁇ -methylleucine); amino acids with extra functional
• Carboxylic acids with a -serine, O-methyl-threonine, etc. amino acids with an extra hydroxy group in the side chain (such as 3-hydroxy-phenylalanine), amino acids with an extra carboxy group (-COOH) in the side chain (3-carboxy-phenylalanine) ), amino acids with an extra S in the side chain (such as ethionine), amino acids whose carboxylic acid functional group in the side chain is protected with an ester (such as aspartic acid-4-methyl ester), and thio groups in the side chain (such as aspartic acid-4-methyl ester).
• an ester such as aspartic acid-4-methyl ester
• thio groups in the side chain such as aspartic acid-4-methyl ester
• VIP2 means "vasoactive intestinal peptide receptor 2", a protein of mammals such as mice, rats, dogs, monkeys, and humans.
• cancer immunity typically means an immune mechanism against cancer cells (tumor cells).
• the host's immune system is able to recognize and eliminate cancer cells.
• Cancer immunity involves not only the adaptive immune system such as cytotoxic T cells (CTL) and humoral immunity, but also innate immunity such as NK cells, NKT cells, macrophages, and granulocytes, which suppress cancer growth. It is known that it can be done.
• CTL cytotoxic T cells
• innate immunity such as NK cells, NKT cells, macrophages, and granulocytes, which suppress cancer growth. It is known that it can be done.
• TAMs tumor-associated macrophages
• Macrophages are a type of phagocyte that originate from the bone marrow and peripheral blood and phagocytose and eliminate invading pathogenic microorganisms and abnormal cells, but macrophages in the cancer microenvironment change their properties depending on that environment and activate host immunity. They are polarized into M1-type macrophages, which have antitumor activity, and M2-type macrophages, which have functions such as suppressing immunity and inflammation, wound healing, tissue remodeling, angiogenesis, and promoting tumor growth. It is known that the M1 type and M2 type are not definitive, and that they shift to each other when stimulated by cytokines (hormones that transmit information between cells).
• cytokines hormones that transmit information between cells.
• the cancer immunity of this embodiment preferably promotes macrophage polarization from M2 type to M1 type.
• the M1 macrophage is selected from the group consisting of INF ⁇ , IL-12, IL-23, TNF ⁇ , IL-6, IL-1, CSCL9, CXCL10, and CXCL11, compared to M2 macrophages. Exhibits increased secretion of inflammatory cytokines and chemokines.
• M1 macrophages have reduced immunosuppressive cytokines and chemokines selected from the group consisting of IL-10, TGF ⁇ , PGE2, CCL2, CCL17, CCL18, CCL22, and CCL24 compared to M2 macrophages.
• M1 macrophages express increased tumor-associated antigens compared to M2 macrophages. In certain embodiments, M1 macrophages increase stimulation of CD8 + T cells and/or NK cells compared to M2 macrophages.
• cyclic peptide A cyclic peptide that is one of the active ingredients of the present invention is disclosed in Patent Document 1, the entire content of which is incorporated into the present application by reference.
• the peptide disclosed in Patent Document 1 is stabilized by bicyclization while maintaining or enhancing the characteristics (pharmacophore) related to the VIPR2 binding activity of VIpep-3 disclosed in Non-Patent Document 1. be. Any of the peptides disclosed in these documents can be used for the new uses of the present invention.
• a cyclic peptide of an embodiment particularly suitable for enhancing cancer immunity has the following formula (1): c[X 1 -Pro 2 -X 3 -Tyr 4 -Leu 5 -Pro 6 -c(X 7 -X 8 -Leu 9 -Cys 10 ]-X 11 )-X 12 -X 13 (1) It consists of an amino acid sequence.
• X 1 represents cysteine, Mpa (3-mercaptopropionic acid) or D-cysteine
• X 3 represents N-methylated glycine, N-methylated alanine, 2-azetidine-2-carboxylic acid.
• X 8 represents tyrosine, proline or arginine
• X 7 and X 11 represent lysine and aspartic acid, ornithine and glutamic acid , represents any combination of aspartic acid and lysine, glutamic acid and ornithine, lysine and glutamic acid, or glutamic acid and lysine
• X 12 and X 13 each independently represent leucine, isoleucine or norleucine
• X 1 and Cys 10 form a disulfide bond between their respective side chains
• X 7 and X 11 form an amide bond between their respective side chains, whereby the peptide of formula (1) has two cyclic structures within the molecule.
• the N-terminal amino group and the C-terminal carboxy group may be modified or deleted.
• a further preferred embodiment of the cyclic peptide is that in the above formula (1), X 1 represents cysteine, X 3 represents proline or serine, X 7 represents lysine, and X 8 represents tyrosine, X 11 represents aspartic acid, and X 12 and X 13 each independently represent leucine, isoleucine or norleucine.
• the N-terminal amino group is acetylated and the C-terminal carboxy group is amidated.
• Examples of the individual cyclic peptides included in the above formula (1) include the following. c(Mpa-Pro-Pro-Tyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH 2 (SEQ ID NO: 1) Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Ile 13 -NH 2 ( SEQ ID NO: 2) and the peptide described in paragraphs 0056 to 0059 of Patent Document 1.
• the peptide according to the present invention has homology in which one to several amino acids are deleted, added, and/or substituted in the amino acid sequence represented by the above formula (1), Those having binding activity are included.
• the number of those amino acids is not particularly limited as long as the peptide has VIPR2 binding activity. However, the number is preferably 1 to 5, more preferably 1 or 2.
• the deletion, addition, and/or substitution location may be at the end or middle of the peptide, and may be at one location or at two or more locations.
• the above amino acid sequence and BLAST Basic Local Alignment Search Tool at the National Center for B iological Information (USA) at least 50%, preferably 70% or more, more preferably 80%, when calculated using the National Center for Biological Information's Basic Local Alignment Search Tool) etc. (for example, using default or initial setting parameters). % or more, particularly preferably 90% or more.
• the peptide according to the present invention also includes various derivatives and/or modifications thereof as long as they solve the problems of the present invention.
• Such derivatives include those in which the saturated fatty chain of the peptide is replaced by an unsaturated fatty chain, those in which some of the atoms of the peptide are replaced with other atoms including radioactive or non-radioactive isotope atoms,
• methoxylation, prenylation, alkylation, etc. e.g., some of the amino groups of the peptide are acetylated, formylated, myristoylated, palmitoylated, pyroglutamylated, alkylated, or deaminated
• peptides examples include, but are not limited to, peptides fused with membrane-permeable peptides, low-molecular-weight compounds, or molecules that induce protein ubiquitination.
• the peptide according to the present invention also includes peptide salts.
• peptide salts salts with physiologically acceptable bases or acids are used, such as addition salts of inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), organic Addition salts of acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide, or alkali , alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), addition salts of amino acids, and the like.
• inorganic acids hydroochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.
• organic Addition salts of acids p-toluene
• the peptide according to the present invention may be a prodrug.
• a prodrug is a compound that is converted into the peptide according to the present invention by a reaction with an enzyme or gastric acid under physiological conditions in a living body, that is, it is converted into the peptide according to the present invention through enzymatic oxidation, reduction, hydrolysis, etc. It refers to a compound that undergoes hydrolysis by gastric acid, etc., and converts into the peptide according to the present invention.
• Prodrugs of the peptide according to the present invention include compounds in which the amino group of the peptide according to the present invention is acylated, alkylated, or phosphorylated (for example, the amino group of the peptide according to the present invention is eicosanoylated, alanylated, pentyl Aminocarbonylated, (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated, or tert-butylated compounds), compounds in which the hydroxy group of the peptide according to the present invention is acylated, alkylated, phosphorylated, or borated (for example, the hydroxy group of the peptide according to the present invention is acetylated, palmitoylated, propanoylated, pivaloylated) , succinylated
• the prodrug of the peptide according to the present invention is one that changes into the peptide according to the present invention under physiological conditions as described in "Molecular Design”, Vol. 7, “Molecular Design”, published by Hirokawa Shoten in 1990, pp. 163-198. It's okay.
• the prodrug may form a salt, and such salts include those exemplified as the salt of the peptide according to the present invention.
• the peptide according to the present invention may be a crystal, and whether it has a single crystal form or a mixture of crystal forms, the peptide according to the present invention is included in the peptide according to the present invention.
• the crystal can be produced by crystallization using a crystallization method known per se.
• the peptide according to the present invention may be a pharmaceutically acceptable co-crystal or co-crystal salt.
• Co-crystal or co-crystal salt refers to two or more types of crystals at room temperature, each having different physical properties (e.g. structure, melting point, heat of fusion, hygroscopicity, solubility, stability, etc.).
• the co-crystal or co-crystal salt can be produced according to a known co-crystallization method.
• composition containing cyclic peptide The cyclic peptide as one of the active ingredients of the present invention, its derivative or modified form can be used in the form of a composition as a medicine, a diagnostic agent, or a research reagent.
• the administration form of the composition is not particularly limited, and may be administered orally or parenterally. Examples of parenteral administration include transmucosal administration (nasal, oral, ophthalmic, pulmonary, vaginal, or rectal administration), injection administration (intravenous injection, subcutaneous injection, intramuscular injection, etc.), and oral administration. Examples include skin administration.
• the peptide in the composition can be subjected to various modifications in view of its tendency to be easily metabolized and excreted.
• biodegradable polymer compounds such as polylactic acid/glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. as sustained release bases. It may also be used as a peptide and encapsulated with a peptide.
• compositions may use the active ingredients as they are, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc.
• dosage forms include liquids (injections, etc.), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, troches, Examples include inhalants, ointments, eye drops, nasal drops, ear drops, poultices, and the like.
• formulations may be immediate release formulations or controlled release formulations such as sustained release formulations (such as sustained release microcapsules).
• Formulation includes, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, and pH adjustment. It can be carried out by a conventional method using appropriate agents, preservatives, antioxidants, etc.
• ingredients used in formulation include purified water, saline, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystals.
• surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, saponin; glycocholic acid, deoxycholic acid, Bile salts such as taurocholic acid; Chelating agents such as EDTA and salicylic acids; Fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; Enamine derivatives, N-acyl collagen peptides, N- Acylamino acids, cyclodextrins, chitosans, nitric oxide donors, etc. may also be used.
• Pills or tablets can also be coated with sugar-coated, gastric-soluble, or enteric-coated substances.
• Injectables can include distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, and the like.
• wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. can be added. If necessary, conventional additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, and wetting agents can also be used in appropriate amounts.
• the composition of the present invention has a cancer immunity-enhancing effect and is therefore particularly suitable for cancer treatment.
• the population of tumor-associated macrophages present in the tumor microenvironment is biased toward the M2 type, it is preferably used to polarize this macrophage population to the M1 type.
• macrophages may be polarized to M1 type outside the body, and the M1 type macrophages may be returned to the body.
• cancer has its common meaning in the art and refers to all types of cancer.
• Non-limiting examples of cancer include carcinomas such as breast cancer, prostate cancer, ovarian cancer, liver cancer, lung cancer, bladder cancer, pancreatic cancer or colon cancer, sarcoma, lymphoma, melanoma, leukemia, germ cell cancer and blastoma. Or adenocarcinoma.
• the cancer may be solid cancer or non-solid cancer, but solid cancer with a developed cancer microenvironment is preferred, and colorectal cancer is particularly preferred.
• cancer immunity enhancement includes, for example, increasing the immune attack power against cancer, restoring the immune attack power that has been braked by cancer (immune checkpoint inhibition therapy, etc.) .
• an immune checkpoint inhibitor may be further used in combination (including inclusion in the composition) in order to further enhance cancer immunity. You can also do that.
• Immune checkpoint inhibitors include, for example, PD-1 inhibitors (nivolumab (Opdivo), pembrolizumab (Keytruda), etc.), CTLA-4 inhibitors (ipilimumab (Yervoy), etc.), PD-L1 inhibitors (avelumab (Bavencio), etc.) , atezolizumab (Tecentriq), avelumab (Bavencio), etc.).
• composition of the present invention may be used in combination with other drugs and treatments useful for the above-mentioned diseases, such as various types of chemotherapy, surgical treatment, and radiotherapy.
• the dosage is determined based on the symptoms, age, sex, and weight of the patient. It varies depending on the sensitivity difference, administration method, administration interval, type of active ingredient, and type of preparation, and is not particularly limited, but for example, 30 ⁇ g to 1000 mg, 100 ⁇ g to 500 mg, 100 ⁇ g to 100 mg can be administered once or in several doses. can do.
• the next amino acid protected with the Fmoc group was treated with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate (HATU)/ Diisopropylethylamine (DIEA) was added to activate it, and the mixture was placed in a column and allowed to react. After the reaction was completed, the product was washed and the Fmoc group was deprotected using 20% piperidine. By repeating this step, the peptide chain was elongated and the Fmoc group of the final amino acid was deprotected, and then the peptide resin was taken out from the apparatus.
• HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate
• DIEA Diisopropylethylamine
• the monocyclic peptide was purified by RT-HPLC using a SunFire C18 column (10 x 150 mm) (manufactured by Waters) and then lyophilized. After dissolving the single-ring peptide in a mixture of Tris-HCl buffer (pH 8.5) and acetonitrile, DMSO was added and stirred at room temperature for 36 hours to cyclize the peptide through a disulfide bond.
• the bicyclic peptide was purified by RT-HPLC using a SunFire C18 column (10 x 150 mm) (manufactured by Waters) and then lyophilized. The molecular weight of the finally obtained peptide was measured using microflex (Bruker), and the target product was identified.
• Table 1 shows the theoretical molecular weight, measured molecular weight, purity, cyclization type, and sequence of the peptide synthesized in this example. Furthermore, among these, the amino acid sequences of Seq-1 and Seq-10 are shown below. In addition, in Table 1 and the following amino acid sequences, amino acids without D-form notation indicate L-form.
• Seq-1 c(Mpa-Pro-Pro-Tyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH 2 (SEQ ID NO: 1)
• Seq-10 Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c (Lys 7 -Tyr 8 -Leu 9 -Cys 10 ] -Asp 11 ) -Leu 12 -Ile 13 -NH 2 (SEQ ID NO: 2)
• Seq-1 and Seq-10 remove the C-terminal 3 residues (Leu-Arg-Ser) of VIpep-3 (see Non-Patent Document 1), and replace the S-S bond between positions 1 and 10 and the 7th position. It is a peptide bicyclized by an amide bond between the -11 position.
• CT-26 colon cancer cells obtained from ATCC were cultured in RPMI (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin.
• FBS fetal bovine serum
• streptomycin 100 IU/mL streptomycin.
• CFP-tagged CT-26 cells Clontech
• pEGFP-C1 plasmid was purified with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's recommendations.
• CT-26 cells were transfected.
• CT-26-GFP cells were selected by cell sorting (Beckman Coulter, Inc.) and treated with 600 ⁇ g/mL geneticin (Thermo Fisher Scientific), 10% FBS, 100 IU/mL penicillin, and 100 IU/mL streptomycin.
• CT-26 cells stably expressing GFP were established by culturing in RPMI containing.
• RAW264.7 cells were cultured in DMEM (manufactured by Sigma-Aldrich) containing 10% FBS, 100 IU/mL penicillin, and 100 IU/mL streptomycin.
• CT-26-CM ⁇ Preparation of CT-26 culture supernatant (CT26-CM)> 4 ⁇ 10 6 CT-26 cells were cultured for 3 days in 20 mL of RPMI medium containing 10% FBS, 100 IU/mL penicillin, 100 IU/mL streptomycin. The supernatant was collected and centrifuged at 400g for 5 minutes. Thereafter, the supernatant was aliquoted and stored at -80°C until use.
• ⁇ RAW264.7 cells cultured in CT26-CM and evaluation of their properties Mouse macrophage RAW264.7 cells (24 ⁇ 10 4 cells) were cultured in the presence of 20% CT26-CM.
• the VIPR1 and VIPR2 antagonist VIPhyb (Gozes I et al. Cell Mol Neurobiol 1995, 15:675-687) (1 ⁇ M and 3 ⁇ M doses) or the selective VIPR2 agonist BAY55-9837 (Tsutsumi et al. M,et al. Diabetes 2002, 51:1453-1460) (100 nM dose) was added.
• RNAimax Lipofectamine RNAimax (manufactured by Thermo Fisher Scientific) according to the manufacturer's recommendations. Three days after culturing CT26-CM, mRNA expression of VIPR1 or VIPR2 was analyzed and knockdown efficiency was evaluated. Expression of M1 and M2 genes was measured 4 days after culturing CT26-CM.
• Relative mRNA expression levels were determined using the 2 - ⁇ Ct method. ⁇ Ct values were calculated using data from the control group. Primer sequences are shown in Table 2 below.
• RAW264.7 cells cultured for 4 days in CT26-CM were labeled with PKH-26 (PKH26 Red Fluorescent Cell Linker Kit, General Cell Membrane Labeling, MERCK, PKH26GL-1KT) (Sigma-Aldrich, manufacturer's instructions). ) at a ratio of 1:2 and co-cultured for 2 hours. Thereafter, RAW264.7 cells were labeled using CD11b-FITC (eBioscience, Thermo Fisher Scientific). The amount of CT26 cells taken up by RAW264.7 cells by phagocytosis was detected using PKH-26. Phagocytosis was analyzed using CytExpert Software (Beckman Coulter) and CytoFLEX (Beckman Coulter).
• CT-26-GFP (1 ⁇ 10 5 cells) was implanted subcutaneously (s.c.) into 6-week-old SCID mice.
• PBS or VIPhyb (10 ⁇ g/day, sc daily, manufactured by KareBay Biochem) was administered for 2 weeks.
• mice were divided into four groups: 1) PBS, 2) VIPhyb (10 ⁇ g/day, daily), 3) anti-PD-1 antibody (200 ⁇ g/day, i. .p., 3 times/week, manufactured by Bio The size of the tumor was measured every week, and the volume of the tumor was calculated as length ⁇ (width ⁇ width)/2.
• Tumors from mice subjected to anticancer tests were removed 12 to 14 days after each treatment and treated with collagenase IV (Sigma-Aldrich), dispase II (Sigma-Aldrich), and DNase I (Thermo Fisher Scientific) in PBS. After enzymatic digestion at 37° C., tumor suspensions were prepared using 100 and 70 ⁇ m cell strainers (Thermo Fisher Scientific). FACS buffer (5% FBS in PBS, 0.1% sodium azide) was added and centrifuged at 400 g at 4°C. Leukocytes were further separated from the tumor suspension by gradient centrifugation at 400 g for 30 minutes at room temperature using Histopaque®-1083 (Sigma-Aldrich), and leukocytes were collected.
• collagenase IV Sigma-Aldrich
• dispase II Sigma-Aldrich
• DNase I Thermo Fisher Scientific
• TILs Tumor-infiltrating leukocytes
• CD16/CD32 eBioscience, Thermo Fisher Scientific
• Antibody information is shown in Table 3 below. The optimal concentration of each antibody was determined before each experiment. Dead cells were stained with LIVE/DEAD TM fixable near-IR dead cell stain kit (Thermo Fisher Scientific). The leukocyte population was defined as follows.
• M1 macrophages CD45 + , CD3e ⁇ , CD11b + , F4/80 + , MHCII high
• M2 macrophages CD45 + , CD3e ⁇ , CD11b + , F4/80 + , MHCII low , CD206 +
• natural killer (NK) cells CD45 + , CD3e ⁇ , CD11b ⁇ , CD335 +
• macrophage phagocytosis of CT-26 CD45 + , CD3e ⁇ , CD11b + , F4/80 + , CT-26-GFP +
• PD-1 + macrophages CD45 + , CD3e ⁇ , CD11b + , PD-1 + ).
• reaction was performed at room temperature for 1 hour using IR-Dye800-conjugated anti-rabbit or anti-mouse secondary antibody (manufactured by LI-COR).
• the membrane was visualized with an Odyssey infrared imaging system (manufactured by LI-COR).
• ⁇ -actin was used as an internal control.
• Colon cancer tissues were fixed with 4% formalin and embedded in paraffin. These tissues were deparaffinized and blocked with 10% goat antiserum for 1 hour at room temperature. Thereafter, it was reacted overnight at 4°C using antibodies against CD68 (1:100 dilution), VIPR1 (1:100 dilution), or VIPR2 (1:100 dilution). Thereafter, the tissue was washed and treated with Alexa flour 488 goat anti-mouse IgG (1:500, Thermo Fisher Scientific) or Alexa flour 594 goat anti-rabbit IgG (1:500, Thermo Fisher Scientific). The following antibodies were used to react in the dark at room temperature for 1 hour. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI) for 10 minutes. Images were taken with a fluorescence microscope (BZ9000; manufactured by Keyence Corporation).
• KS-133> CT-26 (1 ⁇ 10 5 cells) was implanted subcutaneously (s.c.) into 6-week-old Balb/c mice. At the time of tumor formation, mice were divided into four groups: 1) 1% DMSO/PBS, 2) KS-133 (30 ⁇ g/day, sc daily), 3) anti-PD-1 antibody. (200 ⁇ g/day, ip, 3 times/week, manufactured by Bio The size of the tumor was measured every week, and the volume of the tumor was calculated as length ⁇ (width ⁇ width)/2.
• Figure 1A shows the mRNA expression levels of M1 macrophage markers TNF- ⁇ , iNOS, and CXCL10
• Figure 1B shows the mRNA expression levels of M2 macrophage markers Mrc-1, IL-1rn, and CCL-22
• Figure 1C shows the mRNA expression levels of M2 macrophage markers Mrc-1, IL-1rn, and CCL-22
• FIG. 1D shows the mRNA expression levels of immune checkpoint markers SIRP- ⁇ , PD-1, and PD-L1.
• FIG. 1D shows the mRNA expression levels of VIPR1 and VIPR2.
• FIG. 1E shows the protein expression levels of VIPR1 and VIPR2. Data are expressed as mean ⁇ SEM. *: p ⁇ 0.05, **: p ⁇ 0.01 (two-way ANOVA) compared to control.
• CT26-CM significantly reduced the expression of CXCL10 mRNA, an M1 macrophage marker, and had no effect on the expression of TNF- ⁇ and iNOS mRNA.
• M2 macrophage markers mannose receptor (Mrc-1), IL-1rn, and CCL-22 mRNA was significantly increased.
• Figure 1C the expression of immune checkpoint markers SIRP- ⁇ and PD-L1 mRNA was increased, but the expression of PD-1 mRNA was not affected. ( Figure 1C).
• FIG. 2 shows the results that VIPHYb, an antagonist to VIPR1 and VIPR2, induced M1 polarization and promoted phagocytosis of CT-26 by macrophages in RAW264.7 cells cultured in CT26-CM.
• FIG. 2A shows The effect of 1 ⁇ M and 3 ⁇ M VIPhyb on M1 macrophage marker mRNA expression.
• Figure 2B shows the effect on M2 macrophage marker mRNA expression.
• Figure 2C shows the effect on immune checkpoint marker mRNA expression.
• Figure 2D shows RAW264.7 cells themselves (Negative control), RAW264.7 cells that have phagocytosed labeled CT-26 (in the absence of VIPhyb: Control), and RAW264.7 cells that have phagocytosed labeled CT-26.
• Representative FACS histogram of cells in the presence of VIPhyb).
• FIG. 2E shows the results of quantifying and comparing the phagocytosis. Data are expressed as mean ⁇ SEM. *: p ⁇ 0.05 compared to control by two-way ANOVA or Student's t-test.
• VIPhyb a non-selective antagonist for VIPR1 and VIPR2
• M1 macrophage markers TNF- ⁇ , iNOS, and CXCL10 Fig. 2A
• VIPhyb had no effect on the expression of immune checkpoint marker mRNA (Fig. 2C).
• Fig. 2C the phagocytosis of CT-26 cells by RAW264.7 cells treated with VIPhyb enhanced the phagocytosis of CT-26 (FIGS. 2D and E).
• FIG. 3B shows the ratio of M1 macrophages
• FIG. 3C shows the ratio of M2 macrophages
• FIG. 3D shows the ratio of NK cells.
• Figure 3E shows the M1/M2 macrophage ratio
• VIPhyb significantly reduced the growth of subcutaneously implanted tumors in SCID mice compared to controls (Figure 3A).
• the tumor showed a tendency to increase M1 macrophages (FIG. 3B) and a tendency to decrease M2 macrophages (FIG. 3C).
• Fig. 3D no tendency for increase or decrease was observed in NK cells.
• the ratio of M1/M2 macrophages in the TME correlates with patient prognosis. Therefore, when we analyzed the ratio of M1/M2 tumor-associated macrophages, we found that the ratio of M1/M2 tumor-associated macrophages was higher in the VIPhyb-treated group than in the control group ( Figure 3E).
• FIG. 4 shows the results of investigating the combined effect of VIPhyb and anti-PD-1 antibody on the growth of CT-26 tumors in immunodeficient SCID mice.
• FIG. 4B shows the ratio of M1 macrophages
• FIG. 4C shows the ratio of M2 macrophages
• FIG. 4D shows the ratio of NK cells.
• Figure 4E shows the M1/M2 macrophage ratio
• Data are expressed as mean ⁇ SEM. *: p ⁇ 0.05, **: p ⁇ 0.01 compared to control; two-way ANOVA.
• siRNA against VIPR1 did not change the M2 macrophage marker either.
• siRNA against VIPR2 significantly decreased the expression of M2 macrophage markers Mrc-1 and IL-1rn mRNA.
• FIG. 6 shows CD68 (green) and VIPR1 (red, 100x in Figure 6A; 200x in Figure 6B) and CD68 (green) and VIPR2 (red, 100x in Figure 6C) in the tumor stroma of human colon cancer specimens.
• Figure 6D is a representative image of the staining (200x). Nuclei were stained with DAPI (blue).
• the macrophage marker CD68 was highly expressed in the tumor stroma, but almost no CD68-positive cells were seen in the adjacent normal tissue.
• expression of VIPR1 and VIPR2 was observed in cells present in the tumor stroma and the adjacent normal tissue stroma.
• cells expressing CD68 were not positive for VIPR1 staining (FIGS. 6A and 6B) but were positive for VIPR2 staining (FIGS. 6C and 6D).
• FIG. 8 shows the results of VIPR2 selective antagonist KS-133 inducing M1 polarization in RAW264.7 cells cultured in CT26-CM.
• VIPR2 selective antagonist KS-133 showed a tendency to enhance the expression of the M1 macrophage marker iNOS mRNA and significantly enhanced the expression of CXCL10 mRNA (FIG. 8A). Furthermore, the expression of Mrc-1 mRNA, which is one of the M2 macrophage markers, was significantly decreased (Fig. 8B). These data indicate that inhibition of VIPR2 signaling by KS-133 induces macrophage polarization to the M1 type.
• FIG. 10 shows an arrangement of tumors removed from mice after the experiment was completed. In particular, it can be seen that the tumor size was significantly reduced in the group administered with the combination of KS-133 and anti-PD-1 antibody.
• a composition containing a cyclic peptide according to the present invention may be used as a pharmaceutical composition such as a composition for enhancing cancer immunity.

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