WO2023013755A1

WO2023013755A1 - Cancer peptide vaccine cocktail formulation amd method for producing same

Info

Publication number: WO2023013755A1
Application number: PCT/JP2022/030039
Authority: WO
Inventors: 孝行吉森
Original assignee: ブライトパス・バイオ株式会社
Priority date: 2021-08-06
Filing date: 2022-08-05
Publication date: 2023-02-09

Abstract

The present invention addresses the problem of providing a cancer peptide vaccine cocktail formulation, from which a vaccine administration solution can be easily prepared while reducing wasteful disposal of a peptide-containing solution during the preparation of the vaccine administration solution and in which the solubilities of peptides in a solvent are improved, a method for producing the same, etc.　A method for producing a cancer peptide vaccine cocktail formulation, said method comprising step (A) for adding a solvent with pH 2.5-4 to a composition containing a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 2, a peptide of SEQ ID NO: 3 and a peptide of SEQ ID NO. 4.

Description

Cancer peptide vaccine cocktail preparation and method for producing the same

The present invention relates to a cancer peptide vaccine cocktail preparation and a method for producing the same.

Worldwide, approximately 18 million people develop cancer each year, of which approximately 9.6 million die. In Japan, the estimated number of cancer patients in 2019 is about 1 million, and the estimated number of deaths is about 380,000 (https://ganjoho.jp/reg_stat/statistics/stat/short_pred.html). . Surgery, radiation therapy, chemotherapy, molecular-targeted drugs, antibody drugs, immunotherapy, cell therapy, and the like have been developed as major therapeutic methods for such cancers.

Especially in recent years, immune checkpoint inhibitors targeting immune checkpoint molecules typified by CTLA-4, PD-1, and PD-L1 have been developed (Patent Documents 1 and 2). Immunotherapy using these inhibitors has been shown to be able to obtain excellent clinical effects that could not be achieved by conventional treatment methods, and development of various immune checkpoint inhibitors and treatment methods using these is being actively developed. However, immune checkpoint inhibitors do not produce effective clinical effects in all patients, and approximately 20 to 50% of patients do not respond to immune checkpoint inhibitors, and clinical effects are not obtained. There are also cancer types that do not have Therefore, in order to increase the response rate of immune checkpoint inhibitors, combination therapy of such inhibitors and drugs such as chemotherapeutic agents and molecular-targeted drugs is being actively studied.

Cancer vaccines using tumor antigen peptides, which are also used for immunotherapy, are one of the candidate drugs to be used in combination with immune checkpoint inhibitors. Cell-mediated immunity, especially cytotoxic T cells (CTLs) play an important role in the elimination of tumor cells by the body. Precursor T cells that recognize a complex with an antigen (HLA) class I antigen are differentiated and proliferated to attack cancer cells. Therefore, by using a tumor antigen peptide as a drug to induce CTL activity against cancer cells to attack cancer cells, clinical effects such as tumor reduction effects can be obtained.

As such tumor antigen peptides, for example, peptides derived from tumor-specific antigens such as the MAGE-A3 antigen discovered at the Ludwig Institute, EGFRvIII, and gp100, and peptides derived from tumor-associated antigens have been investigated (Patent Documents 3-7, Non-Patent Documents 1-26). However, cancer vaccines containing these peptides alone cannot obtain sufficient clinical effects, and even now there are no cancer vaccines that have been approved. In addition, for example, as a combination therapy with an immune checkpoint inhibitor, a combination trial of a cancer vaccine containing an anti-CTLA-4 antibody and a gp100 peptide was also conducted, but the effect of the combination therapy was not proven. (Non-Patent Document 27).

WO2011/011027 WO2004/004771 WO 99/67288 WO 00/12701 WO 02/010369 WO2009/038026 WO2014/181805

Peptide formulations for vaccines are usually formulated (for example, freeze-dried) for each type of peptide and stored in a container. At a medical institution, it is necessary to prepare a cocktail administration solution by adding water for injection to each container of multiple types of peptide preparations, dissolving them, and mixing the obtained multiple types of peptide-containing liquids to prepare a cocktail administration solution. there were. Moreover, in some cases, an adjuvant is added to such a cocktail administration solution in order to improve the vaccine effect, and the preparation of such a cocktail administration solution becomes more complicated in some cases.
In addition, when preparing the cocktail administration solution as described above, it is necessary to prepare an excessive amount of each peptide-containing solution for the purpose of preventing a shortage of each peptide-containing solution, and as a result, the cocktail When preparing the administration solution, the peptide-containing solution was sometimes left over and wasted.

In addition, among various peptides, there are some that have low solubility in solvents, making it difficult to prepare vaccine administration solutions, and improving the solubility in solvents for such peptides is an issue. Conventionally, sodium hydrogen carbonate has been used to improve the solubility of peptides in aqueous solutions. There are problems that the water content in the freeze-dried preparation cannot be measured accurately, and that the peptide is easily oxidized when it is made into an aqueous solution because the pH becomes basic.

An object of the present invention is to provide a cancer peptide that facilitates the preparation of a vaccine administration solution, reduces wasteful disposal of the peptide-containing solution during preparation of the vaccine administration solution, and improves the solubility of the peptide in a solvent. An object of the present invention is to provide a vaccine cocktail preparation, a method for producing the same, and the like.

Each of the four peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 4 (hereinafter also referred to as "the four peptides of the present invention") has an effect as a cancer peptide vaccine by itself. known until now (Patent Document 6).

In order to solve the above-described problems of the present invention, the present inventors continued to conduct intensive studies on suitable methods for producing cancer peptide vaccine cocktail formulations containing the four types of peptides of the present invention. Furthermore, among the four types of peptides of the present invention, the peptide of SEQ ID NO: 1, in particular, has low solubility in a solvent, and the present inventors diligently studied the conditions of a solvent capable of dissolving such a peptide. As a result of these earnest studies, the present inventors have found the following matters (i) to (iii) and completed the present invention.
(i) The four peptides of the present invention can be dissolved by adding a solvent of pH 2.5 to 4 to the four peptides of the present invention.
(ii) Even if the pH of the peptide solution obtained in (i) above is adjusted to 5.5 to 8, the dissolved state of the peptide can be maintained.
(iii) After drying the peptide solution obtained in (ii) above, water can be added to reconstitute the peptide solution.

That is, the present invention
(1) peptide of Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1);
A peptide of Ala-Ser-Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO: 2);
a peptide of Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO: 3); and
the peptide Leu-Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4);
Step (A) of adding a solvent of pH 2.5 to 4 to the composition comprising
A method for producing a cancer peptide vaccine cocktail formulation comprising;
(2) adding a solvent of pH 2.5 to 4 in an amount such that the concentration of the peptides of SEQ ID NOS: 1 to 4 in the solution obtained in step (A) is 20 mg/mL or less, respectively, to the above (1); the manufacturing method described;
(3) The solution obtained in step (A) is added with a solution of pH 6.5 to 14 and mixed to adjust the pH of the mixed solution to 5.5 to 8 (B). 1) or the production method according to (2);
(4) The production method according to any one of (1) to (3) above, further comprising a step (C) of drying the solution obtained in step (A) or the solution obtained in step (B); and ,
(5) A cancer peptide vaccine cocktail preparation produced by the production method according to any one of (1) to (3) above; and
(6) A cancer peptide vaccine cocktail preparation produced by the production method according to (4) above;
(7) A cancer peptide vaccine cocktail preparation produced by the production method described in (4) above, wherein water is added to prepare a vaccine administration solution, and the vaccine administration solution is administered to a subject. the cancer peptide vaccine cocktail formulation, characterized in that:
etc.

INDUSTRIAL APPLICABILITY According to the present invention, preparation of a vaccine administration solution is simple, wasteful disposal of a peptide-containing solution during preparation of a vaccine administration solution is reduced, and the solubility of the peptide in a solvent is improved. A vaccine cocktail formulation, a method for producing the same, and the like can be provided.
In addition, according to the present invention, cancer peptide vaccine cocktail formulations can be produced at lower manufacturing costs and quality test costs than when the four types of peptides of the present invention are separately formulated. .

FIG. 1 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 1 (glycine buffer, with pH adjustment) under reduced pressure. FIG. 2 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 2 (hydrochloric acid, with pH adjustment) under reduced pressure. FIG. 3 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 3 (hydrochloric acid, no pH adjustment) under reduced pressure. FIG. 4 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 1 (glycine buffer, with pH adjustment) under reduced pressure, then adding 200 μL of water and stirring. FIG. 5 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 2 (hydrochloric acid, pH adjustment) under reduced pressure, adding 200 μL of water, and stirring. FIG. 6 shows a photograph of the tip of the tube after drying the peptide mixture (200 μL) prepared under condition 3 (hydrochloric acid, no pH adjustment) under reduced pressure, adding 200 μL of water, and stirring. FIG. 7 shows that the peptide mixture (200 μL) prepared under condition 1 (glycine buffer, with pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, left to stand for 24 hours, and then the tip of the tube was removed. Observed photographs are shown. FIG. 8 shows that the peptide mixture (200 μL) prepared under condition 1 (glycine buffer, with pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, allowed to stand for 24 hours, and then vortexed. The photograph which observed the tip of a tube is shown. FIG. 9 shows that the peptide mixture (200 μL) prepared under condition 2 (hydrochloric acid, with pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, left to stand for 24 hours, and then the tip of the tube was observed. Shows a photo of FIG. 10 shows that the peptide mixture (200 μL) prepared under condition 2 (hydrochloric acid, with pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, allowed to stand for 24 hours, and then vortexed. The photograph which observed the tip of is shown. FIG. 11 shows that the peptide mixture (200 μL) prepared under condition 3 (hydrochloric acid, no pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, left to stand for 24 hours, and the tip of the tube was observed. Shows a photo of FIG. 12 shows that the peptide mixture (200 μL) prepared under condition 3 (hydrochloric acid, no pH adjustment) was dried under reduced pressure, then 200 μL of water was added, stirred, allowed to stand for 24 hours, and then vortexed. The photograph which observed the tip of is shown. FIG. 13 shows a photograph observed after freeze-drying the peptide mixture in the vial in Test 4 of Example. FIG. 14 shows a photograph observed after adding ultrapure water to the freeze-dried powder in the vial and stirring in Test 4 of Example.

The cancer peptide vaccine cocktail preparation of the present invention (hereinafter also referred to as “the cocktail preparation of the present invention” or simply “the cocktail preparation”) is a composition containing four peptides of the present invention with a pH of 2.5 to 4. is a cancer peptide vaccine cocktail preparation produced by a production method comprising the step (A) of adding a solvent (hereinafter also referred to as “the production method of the present invention”).

The four types of peptides in the present invention are the four types of peptides of SEQ ID NOs: 1 to 4 shown below.
Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1)
Ala-Ser-Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO: 2)
Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO: 3)
Leu-Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4)
These peptides are peptides derived from tumor antigen proteins expressed by cancer cells (herein also referred to as tumor antigen peptides), are recognized by CTLs restricted to HLA class I, and are cytotoxic to cancer cells. It is a peptide that induces activity. As used herein, a cancer peptide vaccine is synonymous with a peptide vaccine for treating cancer, and uses a tumor antigen peptide as an active ingredient to treat cancer by inducing an immune response against cancer cells. Means medicine. As used herein, the term "peptide" is used to include pharmaceutically acceptable salts thereof, unless the context is inappropriate.

"Pharmaceutically acceptable salts" as used herein include acetate, hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, citrate, oxalate , formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, sodium salt, potassium salt, calcium salt, magnesium salts, ammonium salts, triethylammonium salts, triethanolammonium salts, pyridinium salts, diisopropylammonium salts and the like.

Peptides can be produced by conventional methods, for example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vo1.2, Academic Press Inc, New York, 1976; ) 1985; Pharmaceutical Development Series Vol. 14: Peptide Synthesis, Hirokawa Shoten, 1991; Purification and recovery of peptides can be performed by known methods such as chromatography such as gel chromatography, ion column chromatography, affinity chromatography, and fractionation means based on solubility differences using ammonium sulfate, alcohol, or the like. . It is also possible to prepare polyclonal antibodies or monoclonal antibodies specific to these peptides based on information on the amino acid sequences of the peptides, and to specifically adsorb and recover the peptides using the antibodies. The produced peptide is usually recovered as a salt with counterions such as acetic acid, hydrochloric acid, sodium and trifluoroacetic acid in addition to the peptide. Such salts can be used as drug substances in the preparation of cancer peptide vaccine cocktail formulations. When referring to the mass of a peptide herein, it is meant the mass of the peptide in free form, not the salt.

A "composition containing four types of peptides of the present invention" may be a composition consisting of only four types of peptides of the present invention, or, as described later, a composition containing four types of peptides of the present invention and optional ingredients. It may be a composition containing the peptides, or a composition consisting of the four types of peptides and optional ingredients in the present invention. Such a composition may be solid or liquid, but from the viewpoint of peptide stability, it is preferably solid, and more preferably dried.

The mass ratio of the four peptides contained in the composition containing the four peptides of the present invention is not particularly limited. The mass of the peptide of SEQ ID NO: 4 is 0.7 to 1.3: 0.7 to 1.3: 0.7 to 1.3: 0.7 to 1.3, and 0 0.8-1.2: 0.8-1.2: 0.8-1.2: 0.8-1.2: 0.9-1.1: 0.9-1. 1:0.9-1.1:0.9-1.1: is more preferred.
The content of the four types of peptides contained in the above composition may be the same mass for all four types, or may be different.

The "solvent having a pH of 2.5 to 4" in the step (A) of the present specification is not particularly limited as long as it is a solvent having a pH of 2.5 to 4. Inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, and aqueous phosphoric acid Aqueous solution; Organic acid aqueous solution such as citric acid aqueous solution, acetic acid aqueous solution, trifluoroacetic acid aqueous solution, succinic acid aqueous solution; boric acid buffer, citrate buffer, acetate buffer, glycine buffer, sodium carbonate buffer, potassium carbonate buffer, sodium acetate buffer, acetic acid Buffers such as potassium buffer; and from the viewpoint of the solubility of the four types of peptides (especially the peptide of SEQ ID NO: 1) in the present invention, hydrochloric acid is preferred. In one embodiment, the pH 2.5-4 solvent is a pH 2.5-4 solvent other than a citrate buffer, and in another embodiment, the pH 2.5-4 solvent is a pH 2.5-4 solvent other than a glycine buffer, It is a solvent with a pH of 2.5-4.
In addition, when a cocktail preparation is prepared by adding a solvent of pH 2.5 to 4 to a composition containing four types of peptides in the invention, the solvent is a pharmaceutically acceptable pH 2.5 to 4. solvent.

The pH of the "solvent with pH 2.5-4" is not particularly limited as long as it is 2.5-4. 0.5 to 3.5 are preferred.

In step (A), the amount of the "solvent with pH 2.5 to 4" to be added is not particularly limited. The concentration of each of the four peptides of the present invention in the solution obtained by the method is 20 mg/mL or less, preferably 3 mg to 20 mg/mL, more preferably 3 mg to 10 mg/mL, and still more preferably 3 mg to 5 mg/mL. It is preferable that the addition amount is such that The concentrations of the four peptides of the present invention in such a solution may be the same or different.
In step (A), a suitable amount of the "solvent having a pH of 2.5 to 4" is such that the pH of the solution in which the composition containing the four peptides of the present invention is dissolved is adjusted to 2.5. 5 to 4.5, preferably 2.5 to 4.3. Therefore, preferably in the above step (A), a solvent of pH 2.5 to 4 is added to the composition containing the four types of peptides of the present invention to dissolve the peptides, and the pH 2.5 to 4.5 obtaining a solution (preferably pH 2.5 to 4.3).
In step (A), after adding the "solvent having a pH of 2.5 to 4", the mixture is preferably stirred, and more preferably stirred using a vortex mixer.

As the production method of the present invention, in addition to the above-described step (A), "a solution having a pH of 6.5 to 14 is added to the solution obtained in the step (A), mixed, and the pH of the mixed solution is adjusted to 5. It may further include a step (B) of adjusting to 5 to 8, and when used in combination with an adjuvant whose optimum pH is in the neutral range, while maintaining the function of such an adjuvant for a longer period of time, in the present invention From the viewpoint of maintaining the dissolved state of the four types of peptides, it is preferable that the above step (B) is further included. Since the dosing solution for cocktail formulations is usually prepared for multiple people at once, the dissolution state of the peptide must be maintained while maintaining the function of the adjuvant even if the dosing solution is stored for a certain period of time after the preparation of the dosing solution. is preferred.
In addition, when the production method of the present invention includes steps (A) and (B), even the difficult-to-dissolve peptide of SEQ ID NO: 1 is dissolved once by adding a solvent of pH 2.5 to 4. Therefore, even if the pH is adjusted to 5.5 to 8 in the step (B), it is excellent in that the dissolved state of the four peptides of the present invention can be maintained.

The "solvent having a pH of 6.5 to 14" in the step (B) is not particularly limited as long as it is a solvent having a pH of 6.5 to 14. Aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous sodium carbonate solution, aqueous potassium carbonate solution. , sodium acetate aqueous solution, potassium acetate aqueous solution, basic amino acid aqueous solution (preferably an aqueous solution of a basic amino acid selected from histidine, lysine, arginine and tryptophan, more preferably an aqueous solution of histidine); histidine buffer, HEPES buffer, buffers such as sodium phosphate buffer, potassium phosphate buffer, bicarbonate buffer, Tris-HCl buffer;

The pH of the "solvent having a pH of 6.5 to 14" is not particularly limited as long as it is 6.5 to 14, and may be 6.5 to 13 or 6.5 to 12.

In the production method of the present invention, in addition to the above-described step (A), or in addition to the above-described steps (A) and (B), "the solution obtained in the step (A) or in the step (B) A step (C) of drying the obtained solution may be further included, and from the viewpoint of storage stability of the cancer peptide vaccine cocktail preparation, it is preferable to further include the above-described step (C).

As a method for drying the solution obtained in step (A) or the solution obtained in step (B), a conventional method for drying a peptide solution can be used, including drying under reduced pressure and freeze-drying. Freeze-drying is preferred from the viewpoint of ease of dissolution in a solvent (eg, purified water or water for injection).

The production method of the present invention may include any steps other than step (A), steps (A) and (B), or steps (A) to (C).

As such an optional step, specifically, a step (D) of filtering and sterilizing the solution obtained in step (A) or the solution obtained in step (B); and the solution obtained in step (A), step ( Step (E) of filling a container such as a glass vial with the solution obtained in B) or a solution obtained by sterilizing the solution by filtration. In an embodiment, when the production method of the present invention includes the drying step (C) and the filling step (E), the drying step (C) may be performed after the filling step (E), or the drying step (C ) may be followed by the filling step (E), but it is preferable to perform the drying step (C) after the filling step (E).

Other optional steps include the step (F) of adding optional ingredients. Such optional ingredients include buffers, antioxidants, preservatives, excipients, suspending agents, tonicity agents, chelating agents, surfactants, etc. Specifically, phosphoric acid, citric acid , and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, and benzethonium chloride; proteins such as , gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and others including glucose, mannose, and dextrin. chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; , and nonionic surfactants such as polyethylene glycol (PEG). In addition, the aforementioned trehalose and sucrose are suitable as cryoprotectants and osmotic pressure regulators. When the cancer peptide vaccine cocktail formulation contains trehalose and/or sucrose, the content of trehalose and/or sucrose is not particularly limited, but the mass ratio of each peptide to the total mass of trehalose and/or sucrose is 0.01. ~1 or 0.02-0.3 is preferred. When referring to the weight of trehalose or sucrose herein, it means the weight of anhydrous trehalose or anhydrous sucrose.

In an embodiment, when the production method of the present invention has step (F), the order of each step does not matter, and the steps may be performed simultaneously with other steps, but step (A) or step (B) or after step (A) or step (B) and before step (C). Moreover, the step (F) may be performed before the step (A), and for example, the above optional components may be added to the composition containing the four types of peptides of the present invention.

The cocktail preparation produced by the production method of the present invention contains at least four specific peptides (peptides consisting of amino acid sequences shown in SEQ ID NOs: 1 to 4, respectively). The dosage form of the cocktail formulation is not particularly limited, but an aqueous solution formulation or a dry formulation is preferable. From the viewpoint of peptide stability, a dry formulation is preferable. Dry formulations are more preferred.

The cocktail formulation in the present invention is administered in the form of an aqueous solution. When the cocktail formulation is an aqueous solution formulation, it can be administered to the patient as it is, and when it is a dry formulation, it can be dissolved in a pharmaceutically acceptable solvent (e.g., purified water or water for injection) and administered as an aqueous solution. . As a method for such dissolution, it is preferable to add the aforementioned solvent to the dry preparation and then stir the mixture, more preferably stirring using a vortex mixer.

The concentration of each of the four peptides of the present invention in the aqueous solution (i.e., administration solution) at the time of administration of the cocktail preparation of the present invention is not particularly limited, but for example, each is 20 mg/mL or less, preferably 3 mg to 20 mg. /mL, more preferably 3 mg to 10 mg/mL, more preferably 3 mg to 5 mg/mL. Concentrations of the aforementioned four types of peptides in such an administration solution may be the same for all four types, or may be different.

The pH of the aqueous solution (i.e., administration solution) at the time of administration of the cocktail preparation in the present invention is not particularly limited, and examples thereof include pH 2.5 to 8.0. In some cases, when the optimum pH of the adjuvant is in the neutral region, the pH of the above-mentioned administration liquid is 5.5 to 8.0; 5.5 to 7.5; 6.0 to 8.0; and 6.0 to 7.5; and the like are preferred. When preparing the dosing solution of the cocktail formulation, the pH may be adjusted by adding a pH adjuster. More preferably, the administration solution can be prepared by adding and stirring a pharmaceutically acceptable solvent (eg, purified water or water for injection) without adding an adjusting agent.
The cocktail preparation produced by the production method of the present invention including steps (A) to (C) can be prepared using a pharmaceutically acceptable solvent even if no pH adjuster is added when preparing the administration solution. (eg, purified water or water for injection) is added and stirred to adjust the pH to 5.5 to 8.0; 5.5 to 7.5; 6.0 to 8.0; and 6.0 to 7. .5; and the like can be easily prepared. In addition, the cocktail preparation produced by the production method of the present invention is excellent in that it can maintain the dissolved state of the four peptides of the present invention even when the pH of the administration solution is 5.5 to 8.

The turbidity (OD600) of the liquid for administration of the cocktail preparation in the present invention is not particularly limited, but is preferably 0.2 or less, more preferably 0.1 or less, and even more preferably 0.08 or less. A conventional method can be used to measure the turbidity (OD600) of the administration solution.

The type of cancer targeted for the cocktail preparation in the present invention is not particularly limited. For example, cancers include prostate cancer, pancreatic cancer, colon cancer, lung cancer (including non-small cell lung cancer), hematopoietic tumor, brain tumor, uterine cancer, cervical cancer, stomach cancer, melanoma (malignant including melanoma), thyroid cancer, liver cancer, and esophageal cancer. Preferably, the cocktail formulation is used for melanoma (including malignant melanoma) or lung cancer (including non-small cell lung cancer).

The cocktail preparation in the present invention may further contain an adjuvant and may be administered together with the adjuvant. Examples of adjuvants include incomplete Freund's adjuvant (e.g., ISA-51, SEPPIC), polysaccharides such as pullulan, complete Freund's adjuvant, and BCG, which enhance retention of the peptide at the site of administration by emulsifying an aqueous solution of the peptide. , alum, GM-CSF, IL-2, CpG, and the like can be used. The optimum pH of GM-CSF is in the neutral region (pH 6-8), and it is particularly suitable as an adjuvant used in the cocktail formulation of the present invention.

The cocktail preparation in the present invention is usually administered intradermally or subcutaneously to the patient. This is because peptides are rapidly degraded when administered, for example, by intravenous injection, and cannot sufficiently induce an immune response. In addition, antigen-presenting cells that capture antigens, present them on the cell surface via HLA molecules, and activate T cells such as CTLs are present intradermally or subcutaneously. This is because CTLs and the like exhibiting cytotoxic activity can be efficiently activated by doing so. The site of administration is not particularly limited, but it is preferably near the lymph node as close to the cancer lesion as possible from the start of administration, such as the upper arm and thigh. In addition, when inflammation or the like occurs at the site of administration due to a side effect of administration and administration becomes difficult, administration may be performed at other sites (abdomen, etc.).

The cocktail preparation in the present invention is administered to the patient in an amount capable of exhibiting the desired effect (also referred to herein as an "effective amount"). The dosage is not particularly limited as long as intradermal or subcutaneous administration is allowed, but it is preferably 0.1 mg or more, preferably 0.1 mg in terms of mass of dry powder of peptide per peptide. ~3 mg, more preferably 1 mg to 3 mg. The doses of the four types of peptides in the present invention may be the same for all four types, or may be different.

The administration frequency of the cocktail preparation in the present invention may be any frequency as long as an immune response is obtained. Dosing may be once every 2, 3, 4, 5, or 6 weeks, and the dosing frequency may be changed along the way. For example, administration may be performed once a week for 4 times, followed by administration once every 3 to 4 weeks. The number of administrations of the cocktail formulation is, for example, at least 8 times, preferably 16 times or more. There is no upper limit to the number of administrations as long as the patient can tolerate the administration, but there is a track record of up to 84 administrations in clinical trials, and at least this number of administrations is possible.

When the cocktail preparation of the present invention is administered to a patient, CTLs against the administered peptide are activated in the patient's body, cancer cells are eliminated, and clinical effects can be obtained.

The cocktail preparation in the present invention may be used in combination with one or more other antitumor agents or treatment methods. Cancer cells in each patient are a heterogeneous population, and there are cells that cannot be eliminated by immune responses and cells that are resistant to antitumor drugs, hormone therapy, etc. Therefore, the cocktail preparations of the present invention and other Clinical effects such as reduction of cancer lesions and prolongation of survival can be enhanced by combined use of these antitumor drugs and treatment methods.

In certain embodiments, the antineoplastic agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors block the immunosuppressive effects of cancer cells and antigen-presenting cells. Immune checkpoint inhibitors include, for example, agents (e.g., antibodies) against the following molecules: CTLA-4 (ipilimumab, tremelimumab, etc.), PD-1 (nivolumab, pembrolizumab, AMP-224, AMP-514 ( MEDI0680), pidilizumab (CT-011), etc.), LAG-3 (IMP-321, BMS-986016, etc.), BTLA, KIR (IPH2101, etc.), TIM-3, PD-L1 (Durvalumab (MEDI4736), MPDL3280A, BMS -936559, avelumab (MSB0010718C, etc.), PD-L2, B7-H3 (MGA-271, etc.), B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF1 -R. In preferred embodiments, the immune checkpoint inhibitor is an agent against PD-1, such as an anti-PD-1 antibody (nivolumab, pembrolizumab, AMP-514 (MEDI0680), pidilizumab (CT-011), etc.).

Antitumor agents further include alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, microtubule polymerization inhibitors, molecular targeting agents, etc. Specifically, 5-FU, estramustine, Docetaxel, temozolomide, cisplatin, gemzar, rituximab and the like. Treatment methods include surgery, radiation therapy, and hormone therapy (steroids such as dexamethasone, mitoxantrone, presonisolone, estrogen, progesterone, and analogues such as luprin).

As used herein, the term "combination" includes simultaneous or sequential use for the treatment of cancer in the same patient, regardless of the order. When the cocktail preparation of the present invention is used in combination with other anti-tumor drugs or treatment methods, the cocktail preparation activates blood cells such as CTLs and exerts its effect, so it is effective in activating the hematopoietic system and immune response. It is preferable to use other antitumor drugs and treatment methods within the range that does not affect them. For example, administering the cocktail preparation of the present invention after the lymphocyte count (e.g., 1,000 cells/mL or more) recovers after administration of the antitumor agent, or other antitumor drugs after administering the cocktail preparation of the present invention Methods such as using drugs or therapeutic methods, or using other anti-tumor drugs or therapeutic methods within the range that does not cause a decrease in white blood cell count or lymphocyte count during the period of administration of the cocktail preparation of the present invention are conceivable.

In a further aspect, the invention provides a method for treating cancer comprising the peptide of Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1), Ala-Ser - peptide of Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO:2), peptide of Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO:3), and A method comprising administering to a patient a cancer peptide vaccine cocktail formulation comprising the peptide Leu-Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4).

In a further aspect, the present invention provides peptides of Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1), Ala-Ser - peptide of Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO:2), peptide of Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO:3), and Concerning the use of the peptide Leu-Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4).

In a further aspect, the present invention provides a peptide of Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1), Ala-Ser-Leu, for use in the treatment of cancer. - peptide of Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO: 2), peptide of Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO: 3), and Leu- It relates to the peptide Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4).

Although the present invention will be described in detail below with reference to examples, the present invention is not limited to these examples.

As a method for manufacturing cocktail preparations of cancer peptide vaccines, we decided to conduct a specific study on the methods of conditions 1 to 3 in Table 1 below.

[Test 1] Peptide solubility study In order to evaluate the step (i) in Table 1, Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1), Ala- Ser-Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO:2), Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO:3) and Leu-Leu- Using the peptide drug substance of Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4) (manufactured on consignment by Lonza), the solubility of the peptide was examined by the following method.

(Condition 1; Peptide dissolution with glycine buffer)
The peptide drug substance of SEQ ID NO: 1 was added to 50 mM glycine buffer (glycine-HCl buffer) (pH 3.5) and stirred so that the final concentration of the peptide of SEQ ID NO: 1 was 18 mg/mL. The solubility of the peptide in the resulting peptide solution was evaluated by visually observing the appearance of the solution.
Peptides of SEQ ID NOs: 2 to 4 were also added to glycine buffer in the same manner and stirred to evaluate the solubility of the peptides.
Note that the peptide of SEQ ID NO: 1 had to be vortexed for several tens of seconds after being added to the glycine buffer.

Also, 150 μL of the glycine buffer solutions of the peptides of SEQ ID NOS: 1-4 were separated and mixed to prepare 600 μL of a peptide mixture containing the four types of peptides of SEQ ID NOS: 1-4. The solubility of peptides in such a peptide mixture was evaluated by visually observing the appearance of the solution.

(Conditions 2 and 3; Peptide dissolution with 1 mM hydrochloric acid)
The peptide drug substance of SEQ ID NO: 1 was added to 1 mM hydrochloric acid (pH about 3) and stirred so that the final concentration of the peptide of SEQ ID NO: 1 was 18 mg/mL. The peptide solubility in the obtained peptide solution was evaluated.
Peptides of SEQ ID NOs: 2 to 4 were also added to 1 mM hydrochloric acid (pH about 3) in the same manner and stirred to evaluate the solubility of the peptides.
It should be noted that the peptide of SEQ ID NO: 1 could be dissolved more easily when 1 mM hydrochloric acid was used than when glycine buffer was used.

Also, 150 μL of 1 mM hydrochloric acid solutions of the peptides of SEQ ID NOS: 1-4 were dispensed and mixed to prepare 600 μL of a peptide mixture containing the four types of peptides of SEQ ID NOS: 1-4. The solubility of peptides in such a peptide mixture was evaluated by visually observing the appearance of the solution.

(result)
Table 2 shows the results of evaluating the solubility of peptides.

From the results in Table 2, it was found that the individual peptides of SEQ ID NOs: 1 to 4 can be dissolved in an acidic solvent with a pH of 3.5 or 3. In addition, as described above, when 1 mM hydrochloric acid was used (conditions 2 and 3), the peptide could be dissolved with shorter stirring than when 50 mM glycine buffer was used (condition 1). Hydrochloric acid was found to be preferred.
In addition, even when the peptides of SEQ ID NOS: 1 to 4 were mixed, the clear state was maintained. thought to be soluble.
That is, when preparing a cocktail preparation containing a mixture (dry product) of peptides of SEQ ID NOs: 1 to 4, these peptides can be easily and simply dissolved by using an acidic solvent with a pH of 3.5 or 3. It was shown that the administration liquid of the cocktail formulation can be easily and conveniently prepared.

[Test 2] Examination of the effect on peptide solubility by increasing the pH of the peptide mixture 1
When using a cocktail preparation containing a mixture (dried product) of peptides of SEQ ID NOs: 1 to 4, an adjuvant may be further added in order to further enhance the vaccine effect of such a cocktail preparation. There are various types of adjuvants, some of which have an optimum storage pH in the neutral region. Since the dosing solution of the cocktail formulation is usually prepared for a plurality of people at once, it is preferable that the dissolution state of the peptide can be maintained even after the dosing solution is stored for a certain period of time after the dosing solution is prepared.
Therefore, whether or not the dissolved state of the peptides can be maintained even when the pH of the peptide mixture solution is raised to the neutral region (step (ii) in Table 1) was examined by the following method.

(Condition 1; Peptide dissolution with glycine buffer and pH adjustment with histidine buffer)
By the method described in Test 1, Condition 1, peptides of SEQ ID NOs: 1 to 4 were dissolved in 50 mM glycine buffer to prepare 600 μL of peptide mixture. The total concentration of the peptides of SEQ ID NOS: 1-4 in this peptide mixture solution was 18 mg/mL, and the individual peptide concentrations of SEQ ID NOS: 1-4 were each 4.5 mg/mL.
90 μL of 50% sucrose was added to the aforementioned peptide mixture, and the pH was measured. Then, the peptide mixture was adjusted to pH 6 by adding 200 mM histidine buffer (pH 6.5). The pH of the prepared peptide mixture was measured, and the solubility of the peptide was evaluated by visually observing the appearance of the peptide mixture.

(Condition 2; Peptide dissolution with 1 mM hydrochloric acid, and pH adjustment with histidine buffer)
By the method described in Test 1, Condition 2, peptides of SEQ ID NOs: 1 to 4 were dissolved in 1 mM hydrochloric acid to prepare 600 μL of a peptide mixture. The total concentration of the peptides of SEQ ID NOS: 1-4 in this peptide mixture solution was 18 mg/mL, and the individual peptide concentrations of SEQ ID NOS: 1-4 were each 4.5 mg/mL.
90 μL of 50% sucrose was added to the aforementioned peptide mixture, and the pH was measured. Then, the peptide mixture was adjusted to pH 6 by adding 200 mM histidine buffer (pH 6.5). The pH of the prepared peptide mixture was measured, and the solubility of the peptide was evaluated by visually observing the appearance of the peptide mixture.

(result)
Table 3 shows the results of measuring the pH of the peptide mixture and the results of evaluating the solubility of the peptides.

From the results in Table 3, it was found that the peptides of SEQ ID NOS: 1-4 were maintained in a dissolved state even when the pH of the peptide mixture was adjusted to 6.

[Test 3] Confirmation of Peptide Solubility When Water Was Added After Drying the Peptide Mixture After the peptide mixture was dried under reduced pressure, water was added to determine whether the peptide dissolved in water. method.

(Condition 1; Peptide dissolution with glycine buffer and pH adjustment with histidine buffer)
600 μL of peptide mixture was prepared by dissolving peptides of SEQ ID NOs: 1 to 4 in 50 mM glycine buffer by the method described in Test 2, Condition 1, 90 μL of 50% sucrose was added, and then 200 mM histidine buffer (pH 6) was added. .5) was added to adjust the pH to 6, and the liquid volume was adjusted to 900 μL with MilliQ water. The total concentration of the peptides of SEQ ID NOS: 1-4 in the 900 μL peptide mixture was 12 mg/mL, the individual peptide concentrations of SEQ ID NOS: 1-4 were 3.0 mg/mL, and sucrose was 5%. (w/v).
200 μL of this 900 μL peptide mixture was dispensed into three 1.5 mL capacity tubes.

(Condition 2; Peptide dissolution with 1 mM hydrochloric acid, and pH adjustment with histidine buffer)
600 μL of peptide mixture was prepared by dissolving the peptides of SEQ ID NOs: 1 to 4 with 1 mM hydrochloric acid by the method described in Test 2, Condition 2, 90 μL of 50% sucrose was added, and then 200 mM histidine buffer (pH 6.0) was added. 5) was added to adjust the pH to 6, and the liquid volume was adjusted to 900 μL with MilliQ water. The total concentration of the peptides of SEQ ID NOS: 1-4 in the 900 μL peptide mixture was 12 mg/mL, the individual peptide concentrations of SEQ ID NOS: 1-4 were 3.0 mg/mL, and sucrose was 5%. (w/v).
200 μL of this 900 μL peptide mixture was dispensed into three 1.5 mL capacity tubes.

(Condition 3: Peptides are dissolved with 1 mM hydrochloric acid, but pH adjustment with histidine buffer is not performed)
By the method described in Test 1, Condition 3, peptides of SEQ ID NOs: 1 to 4 were dissolved in 1 mM hydrochloric acid to prepare 600 μL of a peptide mixture. After adding 90 μL of 50% sucrose to the peptide mixture, the volume was adjusted to 900 μL with MilliQ water without adjusting the pH. The total concentration of the peptides of SEQ ID NOS: 1-4 in the 900 μL peptide mixture was 12 mg/mL, the individual peptide concentrations of SEQ ID NOS: 1-4 were 3.0 mg/mL, and sucrose was 5%. (w/v).
200 μL of this 900 μL peptide mixture was dispensed into three 1.5 mL capacity tubes.

(Drying under reduced pressure)
The above-mentioned tubes (total of 9 tubes) containing the peptide mixture were placed on an intermediate plate in a desiccator covered with silica gel with the lids opened. Vacuum drying was performed by evacuating the inside of the desiccator to about -0.08 Mpa with a vacuum pump and leaving it for one week.

(result)
Table 4 and FIGS. 1 to 3 show the results of visual observation of the appearance of each tube after drying under reduced pressure.

From the results in Table 4 and FIGS. 1 to 3, in the case of conditions 1 and 2, vacuum drying caused a transparent mass to stick to the bottom of the tube, whereas in the case of condition 3, vacuum drying It was found that a white mass stuck to the bottom of the tube.

In addition, 200 μL of MilliQ water was added to each of the nine dried tubes described above and stirred to reconstitute the peptide mixture. Table 5 and FIGS. 4 to 6 show the results of visual inspection of the appearance of each tube after reconstruction. Table 5 shows the results of measuring the pH and OD600 of the reconstituted peptide mixture.

From the results of Table 5 and FIGS. 4 to 6, it was found that conditions 2 and 3 are more preferable than condition 1 because the dried peptides have higher solubility in water when water is added. . In particular, in the case of conditions 2 and 3, the OD600 value of the solution was particularly low, indicating that the peptide was dissolved particularly well compared to the other cases. In addition, when comparing conditions 2 and 3, the solubility of the peptide was slightly higher under condition 3, in which the pH was not adjusted to a neutral range. It was found that sufficient solubility was obtained.
The pH of the solution when water was added to reconstitute the peptide mixed solution was approximately the same as the pH of the solution before drying under reduced pressure.

In addition, the appearance of each tube after adding MilliQ water and stirring for 24 hours was visually observed. The results are shown in Table 6 and FIGS. Also, after vortexing each of these tubes, the appearance was again visually inspected. These results are shown in Table 6 and FIGS.

From the results in Table 6 and FIGS. 7 to 12, the peptides are more compatible with water under conditions 2 and 3 than under condition 1, even after standing for 24 hours after the addition of water and after vortexing. It was found to be in a dissolved state, which was preferable.

[Test 4] Examination of the effect on peptide solubility by increasing the pH of the peptide mixture 2
In Test 2 above, a 200 mM histidine buffer (pH 6.5) was used as the pH adjuster for increasing the pH of the peptide mixture. In order to investigate, the test was conducted by the following method. In this test, a 200 mM histidine aqueous solution (pH 7.75) was used as a pH adjuster for increasing pH.

(Preparation of each peptide solution)
The peptide drug substance of SEQ ID NO: 1 was added to 1 mM hydrochloric acid (pH about 3) and stirred so that the final concentration of the peptide of SEQ ID NO: 1 was 25 mg/mL.
In addition, the peptide drug substance of SEQ ID NO: 2 was added to 1 mM hydrochloric acid (pH about 3) and stirred so that the final concentration of the peptide of SEQ ID NO: 2 was 25 mg/mL.
In addition, the peptide drug substance of SEQ ID NO:3 was added to 1 mM hydrochloric acid (pH about 3) and stirred so that the final concentration of the peptide of SEQ ID NO:3 was 25 mg/mL.
In addition, the peptide drug substance of SEQ ID NO: 4 was added to 1 mM hydrochloric acid (pH about 3) and stirred so that the final concentration of the peptide of SEQ ID NO: 4 was 25 mg/mL.
Thus, four types of peptide solutions were prepared. When the solubility of the peptide in these four types of peptide solutions was evaluated by visually observing the appearance of the solution, all of the peptide solutions were clear.

(Preparation of peptide mixture)
These four types of peptide solutions, a 50% (w/v) sucrose aqueous solution, a 200 mM histidine aqueous solution (pH 7.75), and ultrapure water were mixed in the amounts shown in Table 7, and SEQ ID NOS: 1-4 were obtained. 1000 μL (pH about 6) of a peptide mixture containing four types of peptides was prepared.

(Freeze-drying of peptide mixture)
After adding 1000 μL (ie, 1 mL) of the peptide mixture to a 3-mL vial, the vial was stoppered with a rubber stopper and frozen at −80° C. in a freezer.

After the temperature of the trap of the freeze dryer reached -30°C or below, the vial was taken out from the freezer, and after removing the rubber stopper, it was placed in the dry chamber of the freeze dryer. After the dry chamber was closed with a cover, the pressure in the dry chamber was started by turning on the pump. The set temperature of the trap during freeze-drying was −45° C. (ie, the initial set temperature of the trap in the freeze-dryer). About 20 hours after the start of depressurization, the pump was turned off and the pressure in the dry chamber was returned to normal pressure to complete freeze-drying. After lyophilization, mixed peptides were obtained in the vials as clumpy white lyophilized powder (white lyophilized cake; FIG. 13).

(Reconstitution of peptide mixture using lyophilisate)
1 mL of ultrapure water was added to the freeze-dried vial. The white lyophilized powder quickly dissolved in ultrapure water and the solution became clear (Fig. 14). No foaming was observed during dissolution.

From the above, it was shown that even when a 200 mM histidine aqueous solution was used as the solution for raising the pH of the peptide mixture, the dissolved state of the peptides could be maintained.

Claims

peptide of Asn-Val-Leu-His-Phe-Phe-Asn-Ala-Pro-Leu (SEQ ID NO: 1);
A peptide of Ala-Ser-Leu-Asp-Ser-Asp-Pro-Trp-Val (SEQ ID NO: 2);
a peptide of Lys-Leu-Lys-His-Tyr-Gly-Pro-Gly-Trp-Val (SEQ ID NO: 3); and
the peptide Leu-Leu-Gln-Ala-Glu-Ala-Pro-Arg-Leu (SEQ ID NO: 4);
Step (A) of adding a solvent of pH 2.5 to 4 to the composition comprising
A method for producing a cancer peptide vaccine cocktail formulation, comprising:
The production method according to claim 1, wherein a solvent of pH 2.5 to 4 is added in an amount such that the concentration of the peptides of SEQ ID NOs: 1 to 4 in the solution obtained in step (A) is 20 mg/mL or less. .
The method according to claim 1, further comprising a step (B) of adjusting the pH of the mixture to 5.5 to 8 by adding and mixing a pH 6.5 to 14 solution to the solution obtained in step (A). manufacturing method.
The production method according to any one of claims 1 to 3, further comprising a step (C) of drying the solution obtained in step (A) or the solution obtained in step (B).
A cancer peptide vaccine cocktail preparation produced by the production method according to any one of claims 1 to 3.
A cancer peptide vaccine cocktail preparation produced by the production method according to claim 4.
A cancer peptide vaccine cocktail preparation produced by the production method according to claim 4, wherein a vaccine administration solution is prepared by adding water, and the vaccine administration solution is administered to a subject, Said cancer peptide vaccine cocktail formulation.