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WO2016047509A1 - Liposome à liaison peptidique, activateur de lymphocyte t cytotoxique, et vaccin antitumoral - Google Patents

Liposome à liaison peptidique, activateur de lymphocyte t cytotoxique, et vaccin antitumoral Download PDF

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Publication number
WO2016047509A1
WO2016047509A1 PCT/JP2015/076214 JP2015076214W WO2016047509A1 WO 2016047509 A1 WO2016047509 A1 WO 2016047509A1 JP 2015076214 W JP2015076214 W JP 2015076214W WO 2016047509 A1 WO2016047509 A1 WO 2016047509A1
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Prior art keywords
peptide
liposome
phospholipid
group
mol
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English (en)
Japanese (ja)
Inventor
大 堀内
俊隆 赤塚
内田　哲也
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Saitama Medical University
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Saitama Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

  • the present invention relates to a cytotoxic T lymphocyte activator and a peptide-bound liposome suitable as an anti-tumor vaccine, a cytotoxic T lymphocyte activator using the peptide-bound liposome, and an anti-tumor vaccine.
  • CTL cytotoxic T lymphocyte
  • TERT Telomere Reverse Transscriptase
  • the amino acid sequence of the antigen protein is analyzed by a computer algorithm, and those having a high predicted affinity with HLA are often selected (see, for example, Non-Patent Document 1).
  • the antigen peptide 540-548 currently undergoing clinical trials is a sequence having the highest predicted affinity for HLA-A2 among the natural sequences of the TERT (for example, Non-patent document 2).
  • tumor antigen-specific cytotoxic T lymphocytes detected from the peripheral blood of tumor patients are in an unresponsive state to antigen stimulation. This is thought to be because, during tumor growth, epitopes that have high affinity with HLA and are easily recognized by the host immune system are presented from tumor cells to CTLs, and immune tolerance is induced.
  • the tumor antigen peptide binds to somatic cell MHC class I, it is presented to the T cell in an inappropriate form lacking costimulation and causes administration-specific immune tolerance. It is possible that there is a possibility.
  • the high affinity epitope of the TERT protein derived from the self-antigen is presented to the T lymphocyte from the tumor cell during the tumor growth process, and as a result, immune tolerance is induced.
  • antigen-specific immune tolerance is induced, antigen-specific T lymphocytes become unresponsive to activation stimuli, and cytotoxic T lymphocytes that exert sufficient anti-tumor effects are induced by anti-tumor vaccines It is expected to be difficult to do.
  • an anti-tumor vaccine capable of causing a cellular immune response without inducing immune tolerance has not been developed yet, and its prompt provision is strongly demanded.
  • the present invention relates to a peptide-bound liposome capable of inducing a cellular immune response without inducing immune tolerance, a cytotoxic T lymphocyte activator using the peptide-bound liposome, and an antitumor vaccine
  • the purpose is to provide.
  • Means for solving the problems are as follows. That is, ⁇ 1> A peptide-bonded liposome characterized in that a peptide comprising the amino acid sequence represented by SEQ ID NO: 14 is bound to the surface of the liposome. ⁇ 2> A cytotoxic T lymphocyte activator comprising the peptide-bonded liposome according to ⁇ 1>. ⁇ 3> An antitumor vaccine comprising the peptide-bonded liposome according to ⁇ 1>.
  • FIG. 1A is a diagram showing the results when antigen stimulation was performed with epitope peptide # 1 in Test Example 2.
  • FIG. 1B is a diagram showing the results when antigen stimulation was performed with epitope peptide # 2 in Test Example 2.
  • FIG. 1C is a diagram showing the results when antigen stimulation was performed with epitope peptide # 3 in Test Example 2.
  • FIG. 1D is a diagram showing the results when antigen stimulation was performed with epitope peptide # 4 in Test Example 2.
  • FIG. 1E is a diagram showing the results when antigen stimulation was performed with epitope peptide # 5 in Test Example 2.
  • FIG. 1F is a diagram showing the results when antigen stimulation was performed with epitope peptide # 6 in Test Example 2.
  • FIG. 1A is a diagram showing the results when antigen stimulation was performed with epitope peptide # 1 in Test Example 2.
  • FIG. 1B is a diagram showing the results when antigen stimulation was performed with epitope peptide # 2 in Test Example 2.
  • FIG. 1G is a diagram showing the results when antigen stimulation was performed with epitope peptide # 7 in Test Example 2.
  • FIG. 1H is a diagram showing the results of antigen stimulation with epitope peptide # 8 in Test Example 2.
  • FIG. 1I is a diagram showing the results of antigen stimulation with epitope peptide # 9 in Test Example 2.
  • FIG. 1J is a diagram showing the results when antigen stimulation was performed with epitope peptide # 10 in Test Example 2.
  • FIG. 1K is a diagram showing the results when antigen stimulation was performed with epitope peptide # 11 in Test Example 2.
  • FIG. 1L is a diagram showing the results of antigen stimulation with epitope peptide # 12 in Test Example 2.
  • 1M is a diagram showing the results when antigen stimulation was performed with epitope peptide # 13 in Test Example 2.
  • FIG. 1N is a diagram showing the results of antigen stimulation with epitope peptide # 14 in Test Example 2.
  • FIG. 10 is a diagram showing the results when antigen stimulation was performed with epitope peptide # 15 in Test Example 2.
  • FIG. 1P is a diagram showing the results when antigen stimulation was performed with epitope peptide # 16 in Test Example 2.
  • FIG. 1Q is a diagram showing the results when antigen stimulation was performed with epitope peptide # 17 in Test Example 2.
  • FIG. 1R is a diagram showing the results of antigen stimulation with epitope peptide # 18 in Test Example 2.
  • FIG. 1S is a diagram showing the results when antigen stimulation was performed with epitope peptide # 19 in Test Example 2.
  • FIG. 1T is a diagram showing the results of antigen stimulation with epitope peptide # 20 in Test Example 2.
  • FIG. 2A is a diagram showing the results of antigen stimulation with an epitope peptide corresponding to the epitope peptide used for immunization in Test Example 3.
  • FIG. 2B is a diagram showing the results of antigen stimulation with the natural epitope peptide of the epitope peptide corresponding to the epitope peptide used for immunization in Test Example 3.
  • FIG. 3 is a diagram showing the results of Test Example 4.
  • FIG. 4 is a diagram showing the results of Test Example 5.
  • FIG. 5 is a diagram showing the results of measuring the area of the tumor 24 days after transplantation in Test Example 6.
  • FIG. 6 is a diagram showing the results of examining the survival time of mice in Test Example 6.
  • peptide-bonded liposome In the peptide-bonded liposome of the present invention, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is bound to the surface of the liposome, and further contains other components as necessary.
  • the peptide in the peptide-bonded liposome has an amino acid sequence represented by SEQ ID NO: 14 below.
  • LQAYRFHAV SEQ ID NO: 14
  • the amino acid sequence represented by SEQ ID NO: 14 is obtained by substituting the 952nd amino acid with V in the amino acid sequence corresponding to the 944th to 952nd positions in the human TERT sequence.
  • the peptide When used in a state bound to the surface of the liposome, the peptide is specifically taken up by professional antigen-presenting cells, cross-presented to major histocompatibility complex (MHC) class I, and provided with appropriate stimulation
  • MHC major histocompatibility complex
  • the peptide (hereinafter also referred to as “antigen” or “epitope peptide”) is cytotoxic T lymphocytes restricted to HLA-A2 (also referred to as “HLA-A * 0201”), and Cytotoxic T lymphocytes restricted to HLA-A24 (sometimes referred to as “HLA-A * 2402”) may be induced.
  • the term “inducing cytotoxic T lymphocytes” refers to the number of cytotoxic T lymphocytes that specifically recognize the antigen in the living body of the mammal when the mammal is immunized with the antigen. It means that at least one of the activities increases.
  • cytotoxic T lymphocyte There is no restriction
  • the peptide can induce cytotoxic T lymphocytes and activate cellular immunity.
  • the peptide may be designed so as to be cleaved by the action of a proteasome or the like in a cell. Further, the peptide may have a residue as an additional sequence that can overcome selection of a presentation antigen due to individual differences (polymorphism) of MHC when presented on MHC in a cell.
  • a well-known method can be selected suitably, For example, the method of manufacturing by chemical synthesis, the method of manufacturing by biosynthesis, etc. are mentioned. Examples of the method for producing by chemical synthesis include liquid phase synthesis and solid phase synthesis. Examples of the production method by biosynthesis include a method of culturing a transformant introduced with an expression vector capable of expressing the peptide and isolating the peptide from the culture by a well-known purification technique such as an affinity column. Is mentioned. The method for constructing the expression vector is not particularly limited, and a known method can be appropriately selected.
  • the expression vector is constructed by linking a polynucleotide encoding the peptide downstream of a promoter in an appropriate expression vector.
  • an appropriate expression vector can do.
  • the liposome in the peptide-bonded liposome is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose.
  • the phospholipid membrane constituting the liposome has a structure in which a phospholipid, which is an amphiphilic surfactant, forms an interface with a polar group facing the aqueous phase, and a hydrophobic group faces the opposite side of the interface.
  • the liposome refers to a phospholipid bilayer membrane having a closed space.
  • the phospholipid membrane constituting the liposome is a phospholipid having any one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond. And a liposome stabilizer, and an embodiment containing other components as required is preferred.
  • the phospholipid may be composed of a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond, or may be carbonized having 14 to 24 carbon atoms having one unsaturated bond. It may be composed of a phospholipid having a hydrogen group, or may be composed of both. Among these, it has an acyl group having 14 to 24 carbon atoms having one unsaturated bond in that cytotoxic T lymphocytes (CD8 + T cells, CTL) can be more efficiently and specifically enhanced. It preferably consists of phospholipids.
  • the unsaturated acyl group or unsaturated hydrocarbon group bonded to the 1-position and 2-position of the glycerin residue of the phospholipid may be the same or different. From the viewpoint of productivity, it is preferable that they are the same.
  • the number of carbon atoms of the acyl group is not particularly limited as long as it is 14 to 24, and can be appropriately selected according to the purpose, but is preferably 16 to 22, more preferably 18 to 22, and particularly preferably 18. .
  • the carbon number of the acyl group is less than 13, the stability of the liposome may deteriorate, or the CTL activity enhancing effect may be insufficient.
  • the carbon number of the acyl group exceeds 24, Liposomes may become less stable.
  • Specific examples of the acyl group include palmitooleoyl group, oleoyl group, and elcoyl group. Among these, an oleyl group is preferable.
  • the number of carbon atoms of the hydrocarbon group is not particularly limited as long as it is 14 to 24, and can be appropriately selected according to the purpose, but is preferably 16 to 22, more preferably 18 to 22, and particularly preferably 18. preferable.
  • Specific examples of the hydrocarbon group include a tetradecenyl group, a hexadecenyl group, an octadecenyl group, a C20 monoene group, a C22 monoene group, and a C24 monoene group.
  • said acidic phospholipid there is no restriction
  • the acidic phospholipids the number of carbons having one unsaturated bond in terms of the ability to enhance the CTL activity to a practically sufficient level, the industrial supplyability, the quality for use as a pharmaceutical, and the like.
  • Diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid and diacylphosphatidylinositol having 14 to 24 acyl groups are preferred.
  • the acidic phospholipid imparts an anionic ionizing group to the surface of the liposome, and thus imparts a negative zeta potential to the surface of the liposome. For this reason, liposomes have a charge repulsive force and can exist as a stable preparation in an aqueous solvent.
  • the acidic phospholipid is important in ensuring the stability of the liposome when the peptide-bound liposome is in an aqueous solvent.
  • the neutral phospholipid there is no restriction
  • the neutral phospholipid has a higher function of stabilizing the liposome and can improve the stability of the membrane as compared with the phospholipid combined with the acidic phospholipid and the peptide. Therefore, the phospholipid membrane preferably contains neutral phospholipid.
  • the reactive phospholipid is a phospholipid having a functional group to which the peptide can bind.
  • the reactive phospholipid is not particularly limited and may be appropriately selected depending on the intended purpose. However, the reactive phospholipid may be appropriately selected according to the purpose, and is an acyl group having 14 to 24 carbon atoms having one unsaturated bond and carbon having one unsaturated bond.
  • a phospholipid having any of the hydrocarbon groups of formulas 14 to 24 is preferred. These may be used individually by 1 type and may use 2 or more types together. There is no restriction
  • the reactive phospholipid examples include phosphatidylethanolamine or a terminal modified product thereof, phosphatidylglycerol or a terminal modified product thereof, phosphatidylserine or a terminal modified product thereof, phosphatidic acid or a terminal modified product thereof, phosphatidylinositol or a terminal modified product thereof.
  • examples include the body.
  • phosphatidylethanolamine or a terminally modified product thereof is preferable from the viewpoint of industrial availability, simplicity of the coupling step with the peptide, yield, and the like.
  • the phosphatidylethanolamine has an amino group capable of binding the peptide at its end.
  • CTL activity can be enhanced to a practically sufficient level, stability in liposomes, industrial supplyability, quality for use as a pharmaceutical, and the like.
  • Particularly preferred is diacylphosphatidylethanolamine having a C 14-24 acyl group having one unsaturated bond or a terminally modified product thereof.
  • a well-known method can be selected suitably, For example, a base exchange reaction of choline and ethanolamine is carried out using phospholipase D from diacyl phosphatidylcholine as a raw material. Can be manufactured. Specifically, a chloroform solution in which diacylphosphatidylcholine is dissolved and water in which phospholipase D and ethanolamine are dissolved can be mixed at an arbitrary ratio to obtain a crude reaction product.
  • the crude reaction product can be purified on a silica gel column using a chloroform / methanol / water solvent to obtain the desired diacylphosphatidylethanolamine.
  • Column purification conditions such as the composition ratio of the solvent are not particularly limited and may be appropriately selected depending on the purpose.
  • the end-modified product of diacylphosphatidylethanolamine is not particularly limited and may be appropriately selected depending on the purpose.
  • one end of a divalent reactive compound is bonded to the amino group of diacylphosphatidylethanolamine.
  • diacylphosphatidylethanolamine terminal-modified products are not particularly limited and may be appropriately selected depending on the purpose.
  • one end of a divalent reactive compound is bonded to the amino group of diacylphosphatidylethanolamine.
  • diacylphosphatidylethanolamine terminal-modified products are examples of diacylphosphatidylethanolamine.
  • the divalent reactive compound is not particularly limited and may be appropriately selected depending on the intended purpose.
  • at least one of an aldehyde group or a succinimide group capable of reacting with the amino group of diacylphosphatidylethanolamine is used.
  • Specific examples of the divalent reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, terephthalaldehyde and the like. Among these, glutaraldehyde is preferable.
  • divalent reactive compound having a succinimide group examples include dithiobis (succinimidyl propionate), ethylene glycol-bis (succinimidyl succinate), disuccinimidyl succinate, and disuccinimid. Examples include jilsberate and disuccinimidyl glutarate.
  • a divalent reactive compound having a succinimide group at one end and a maleimide group at the other end for example, N-succinimidyl 4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (p- Maleimidophenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4- (p-maleimidophenyl) propionate, succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate Sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- ( ⁇ -maleimidobutyryloxy) succinimide, N- ( ⁇ -maleimidocaproyloxy) succinimide, and the like
  • a terminal modified form of diacylphosphatidylethanolamine can be obtained by bonding a functional group at one end of the above divalent reactive compound to the amino group of diacylphosphatidylethanolamine.
  • the phospholipid membrane is a kind of phospholipid having any one of the acyl group having 14 to 24 carbon atoms having one unsaturated bond and the hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond.
  • two or more types are included, more preferably three or more types are included.
  • the phospholipid includes at least one unsaturated bond selected from diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine. It is preferable to contain a phospholipid having any one of an acyl group having 14 to 24 carbon atoms having 1 and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond. As for the kind of the said phospholipid, 2 or more types are more preferable, and 3 or more types are especially preferable.
  • the phospholipid membrane is acidic (1) any one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond.
  • the phospholipid membrane is capable of stably binding the peptide to the liposome via the functional group of the peptide, and thus an amino group, succinimide group, maleimide group, thiol group, carboxyl group, hydroxyl group, disulfide group
  • it preferably has a functional group such as a hydrophobic group composed of a hydrocarbon group (an alkyl group or the like) having a methylene chain.
  • a hydrophobic group composed of a hydrocarbon group (an alkyl group or the like) having a methylene chain.
  • an amino group, a succinimide group, and a maleimide group are more preferable.
  • Liposome stabilizer-- There is no restriction
  • the other components are not particularly limited as long as liposomes can be formed, and known components can be appropriately selected.
  • composition of the phospholipid membrane examples include the following.
  • B Liposome stabilizer: 0.2 mol% to 75 mol%
  • content of each component is mol% with respect to all the structural components of the phospholipid membrane which comprises the liposome part of a peptide bond liposome.
  • the content of the component (A) is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the content of the component (B) is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of liposome stability, 5 mol% to 70 mol% is preferable, and 10 mol% to 60 mol% is more preferable, and 20 mol% to 50 mol% is particularly preferable. When content of the said component (B) exceeds 75 mol%, the stability of a liposome may be impaired.
  • the component (A) includes the following.
  • the content of the component (a) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.01 mol% to 85 mol%, preferably 0.1 mol% to 80 mol%.
  • the content of the component (b) is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 0.2 mol% to 80 mol%, preferably 0.3 mol% to 60 mol%. More preferably, it is 0.4 mol% to 50 mol%, more preferably 0.5 mol% to 25 mol%.
  • the content of the component (b) is less than 0.2 mol%, the amount of the peptide decreases, and it becomes difficult to activate cytotoxic T lymphocytes to a practically sufficient level. In some cases, if it exceeds 80 mol%, the stability of the liposome may decrease.
  • the phospholipid of component (a) includes the acidic phospholipid and the neutral phospholipid.
  • the phospholipid of component (b) includes the reactive phospholipid.
  • the content of the acidic phospholipid is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 mol% to 85 mol%, more preferably 2 mol% to 80 mol%, and more preferably 4 mol% % To 60 mol% is more preferable, and 5 mol% to 40 mol% is particularly preferable.
  • the content of the acidic phospholipid is less than 1 mol%, the zeta potential is decreased, the stability of the liposome is lowered, and it is difficult to activate cytotoxic T lymphocytes to a practically sufficient level. It may become.
  • the content of the acidic phospholipid exceeds 85 mol%, the content of the phospholipid bound to the peptide of the liposome is decreased, and the cytotoxic T lymphocyte is activated to a practically sufficient level. May be difficult to do.
  • the content of the neutral phospholipid is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.01 mol% to 80 mol%, preferably 0.1 mol% to 70 mol%. More preferably, 0.1 mol% to 60 mol% is still more preferable, and 0.1 mol% to 50 mol% is particularly preferable.
  • the content of the neutral phospholipid exceeds 80 mol%, the content of acidic phospholipids, peptide-bound phospholipids and liposome stabilizers contained in the liposomes decreases, and the cells are at a practically sufficient level. It may be difficult to activate injured T lymphocytes.
  • the phospholipid bound to the peptide is obtained by binding the peptide to the reactive phospholipid.
  • the ratio of the reactive phospholipid binding to the peptide is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the type of functional group used for binding, the binding treatment conditions, and the like.
  • a terminal modified form of diacylphosphatidylethanolamine obtained by binding one end of disuccinimidylsuccinate a divalent reactive compound, to the terminal amino group of diacylphosphatidylethanolamine is used as a reactive phospholipid.
  • 10% to 99% of the reactive phospholipid can be bound to the peptide. In this case, the reactive phospholipid not bound to the peptide becomes acidic phospholipid and is contained in the liposome.
  • compositions are mentioned, for example.
  • Acidic phospholipid having one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond 1 mol% ⁇ 85 mol%
  • Neutral phospholipid having any one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond.
  • the following composition may be mentioned.
  • Component (I) 2 mol% to 80 mol%
  • Component (II) 0.1 mol% to 70 mol%
  • Component (III) 0.3 mol% to 60 mol%
  • Component (IV) 10 mol% to 70 mol%. (Total 100 mol%)
  • the following composition can be mentioned.
  • the following composition can be mentioned.
  • the phospholipid membrane may contain a phospholipid containing either an unsaturated acyl group having less than 14 or more than 24 carbon atoms and an unsaturated hydrocarbon group, as long as the effects of the present invention are not impaired.
  • Ratio of the number of unsaturated acyl groups or unsaturated hydrocarbon groups having 14 to 24 carbon atoms to the total number of all unsaturated acyl groups or unsaturated hydrocarbon groups contained in the phospholipid in the phospholipid membrane Is not particularly limited and can be appropriately selected according to the purpose, but is preferably 50% or more, more preferably 60% or more, further preferably 75% or more, particularly preferably 90% or more, and 97% or more. (Eg substantially 100%) is most preferred.
  • the phospholipid membrane may contain lipids other than phospholipids having an acyl group or hydrocarbon group having 14 to 24 carbon atoms as long as the effects of the present invention are not impaired.
  • the content of lipids other than phospholipids having an acyl group or hydrocarbon group having 14 to 24 carbon atoms is not particularly limited and may be appropriately selected depending on the intended purpose. Preferably, 20 mol% or less is more preferable, 10 mol% or less is still more preferable, and 5 mol% or less (for example, substantially 0 mol%) is especially preferable.
  • a well-known method can be selected suitably. For example, extrusion method, vortex mixer method, ultrasonic method, surfactant removal method, reverse phase evaporation method, ethanol injection method, prevesicle method, French press method, W / O / W emulsion method, annealing method, freeze thawing Law.
  • the said liposome there is no restriction
  • the liposome of the said form can be manufactured by selecting the manufacturing method of the said liposome suitably.
  • the particle size of the liposome is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of storage stability, 20 nm to 600 nm is preferable, 30 nm to 500 nm is more preferable, and 40 nm to 400 nm is preferable. More preferred is 50 nm to 300 nm, and most preferred is 70 nm to 230 nm.
  • the particle size of the liposome can be measured by a dynamic light scattering method.
  • a saccharide or a polyhydric alcohol is added to or dissolved in at least one of the internal aqueous phase and the external aqueous phase of the liposome after or during the preparation of the liposome. Also good.
  • a saccharide or polyhydric alcohol is added or dissolved as a liposome protective agent, and water is removed by lyophilization. It is preferable to do.
  • the saccharide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; saccharose, lactose, cellobiose, trehalose, maltose Disaccharides such as raffinose and melezitose; oligosaccharides such as cyclodextrin; polysaccharides such as dextrin; sugar alcohols such as xylitol, sorbitol, mannitol and maltitol. These may be used individually by 1 type and may use 2 or more types together. Among the saccharides, monosaccharides and disaccharides are preferable, and glucose and sucrose are more preferable in terms of availability.
  • the polyhydric alcohol is not particularly limited and may be appropriately selected depending on the intended purpose.
  • glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, octaglycerin, nona Glycerin compounds such as glycerin, decaglycerin, polyglycerin; sugar alcohol compounds such as sorbitol and mannitol; ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene
  • examples include glycol and nonaethylene glycol.
  • polyhydric alcohols glycerin, diglycerin, triglycerin, sorbitol, mannitol, and polyethylene glycol having a molecular weight of 400 to 10,000 are preferable from the viewpoint of availability.
  • the concentration of the saccharide or polyhydric alcohol to be contained in at least one of the inner aqueous phase and the outer aqueous phase of the liposome is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the mass concentration is preferably 1% by mass to 20% by mass, and more preferably 2% by mass to 10% by mass.
  • the peptide is bound to the surface of the liposome.
  • the form of the bond is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the peptide includes an acyl group having 14 to 24 carbon atoms having one unsaturated bond, and an unsaturated bond.
  • An embodiment in which one is bonded to a phospholipid having any one of the hydrocarbon groups having 14 to 24 carbon atoms is preferred.
  • the peptide can be bound to the surface of the liposome via a functional group of the peptide.
  • bonding to the surface of the said liposome According to the objective, it can select suitably, For example, an amino group, a thiol group, a carboxyl group, a hydroxyl group, a disulfide group And a hydrophobic group composed of a hydrocarbon group (alkyl group or the like) having a methylene chain.
  • the amino group, thiol group, carboxyl group, hydroxyl group, and disulfide group can bind the peptide to the surface of the liposome through a covalent bond.
  • the amino group and the carboxyl group can bind the peptide to the surface of the liposome by ionic bond.
  • the hydrophobic group can bind the peptide to the surface of the liposome by a hydrophobic bond between hydrophobic groups.
  • a mode of binding to the surface of the liposome via an amino group, a carboxyl group, or a thiol group is preferable.
  • the ionic bond and the hydrophobic bond are preferable from the viewpoint of ease of preparation of peptide-bonded liposomes because the peptide binding procedure to the liposome is simple.
  • the covalent bond is preferable from the viewpoint of the peptide binding stability on the liposome surface and the storage stability when the peptide-bonded liposome is put into practical use.
  • One feature of the peptide-bonded liposome is that a peptide having an excellent activating effect on cytotoxic T lymphocytes is bound to the surface of the liposome as a constituent component thereof.
  • the peptide is stably bound to the surface of the liposome even after being administered into the living body, for example, by an injection action.
  • the bond between the peptide and the liposome is preferably a covalent bond.
  • the combination of the functional group possessed by the peptide and the functional group possessed by the phospholipid membrane constituting the liposome is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose.
  • the method for binding the peptide to the surface of the liposome is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a liposome containing the reactive phospholipid is prepared, and then the peptide is added.
  • a method of binding a peptide to a reactive phospholipid of a liposome can be mentioned.
  • bonding method of the said peptide to the said reactive phospholipid A well-known method can be selected suitably.
  • the peptide-bonded liposome can be easily produced by preparing a liposome before binding a peptide and then binding the peptide.
  • a suspension of the liposome containing the phospholipid, the liposome stabilizer, and the reactive phospholipid for binding the peptide to the membrane surface is prepared, and one of the sugars is added to the outer aqueous phase. Add about 2% to 10% by weight of sucrose and dissolve.
  • the saccharide additive is transferred to a 10 mL glass vial, placed in a shelf-type lyophilizer, frozen at ⁇ 40 ° C. or the like, and then freeze-dried liposomes can be obtained by a conventional method.
  • the lyophilized product of the liposome can be stored for a long period of time because the water has been removed.
  • the peptide-bound liposome can be easily and rapidly added by adding a specific peptide when necessary and performing the subsequent steps. Obtainable.
  • the interaction between the peptide and the liposome is strong and unstable, it is very convenient to store it at the lyophilized product of the liposome and bind the peptide when necessary. .
  • the phospholipid membrane constituting the liposome part of the peptide-bonded liposome can have a phospholipid to which a peptide is bound.
  • the method for producing the liposome containing the phospholipid bound to the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include the following methods (i) and (ii). . (I) Reactivity contained in the liposome by preparing a liposome containing the phospholipid, the reactive phospholipid, and the liposome stabilizer, and adding the peptide and the divalent reactive compound thereto. A method of linking a functional group of a phospholipid and a functional group of the peptide via a divalent reactive compound.
  • a liposome containing a phospholipid, a reactive phospholipid, and a liposome stabilizer is prepared, and a peptide is added thereto, and the functional group of the reactive phospholipid contained in the liposome and the functional group of the peptide A method of connecting and joining.
  • the divalent reactive compound that can be used in the method (i) can be the same as that used in the preparation of the terminal modified product of the reactive phospholipid.
  • Specific examples of the divalent reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, and terephthalaldehyde. Among these, glutaraldehyde is preferable.
  • N-succinimidyl-4- (p-maleimidophenyl) butyrate N-succinimidophenyl butyrate
  • sulfosuccinimidyl-4- (p-maleimide) Phenyl) butyrate N-succinimidyl-4- (p-maleimidophenyl) acetate
  • succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate examples include sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- ( ⁇ -maleimidobutyryloxy) succinimide, N- ( ⁇ -maleimidocaproyloxy) succinimide,
  • the peptide can be bound to the reactive phospholipid contained in the phospholipid membrane constituting the liposome, and the liposome bound with the peptide is formed.
  • a specific example of the method of binding the liposome as a raw material and the peptide via a divalent reactive compound is, for example, a method using a Schiff base bond.
  • the method for binding the liposome and the peptide via the Schiff base bond is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a liposome having an amino group on its surface is prepared, and the peptide is added to the peptide.
  • Examples thereof include a method in which a dialdehyde is added as a divalent reactive compound after being added to a liposome suspension, and an amino group on the liposome surface and an amino group in the peptide are bound via a Schiff base.
  • a reactive phospholipid having a functional group capable of forming an amide bond, a thioether bond, a Schiff base bond, an ester bond or the like is introduced into the phospholipid membrane constituting the liposome.
  • the method of doing is mentioned.
  • Specific examples of the functional group include succinimide group, maleimide group, amino group, imino group, carboxyl group, hydroxyl group, and thiol group.
  • the reactive phospholipid introduced into the phospholipid membrane constituting the liposome include the acyl group having 14 to 24 carbon atoms having one unsaturated bond and the carbon number having 14 to 14 carbon atoms having one unsaturated bond.
  • examples thereof include a terminal modified product of an amino group terminal of a reactive phospholipid having any of 24 hydrocarbon groups (for example, phosphatidylethanolamine).
  • a Schiff base bond can be formed by reacting with an succinimide group.
  • a thiol group is intended, a thioether bond can be formed by reacting with a maleimide group.
  • the peptide in the peptide-bonded liposome of the present invention is considered to be a low immunogenic antigen that has a low BIMAS score and is not recognized by the immune system in vivo as shown in the test examples described later. Therefore, since the peptide-bonded liposome of the present invention can induce a cellular immune response without inducing immune tolerance, the cytotoxic T lymphocyte activator of the present invention, which will be described later, and an antitumor vaccine, It can be suitably used.
  • the cytotoxic T lymphocyte activator of the present invention contains at least the peptide-bonded liposome of the present invention, and further contains other components as necessary.
  • the antitumor vaccine of the present invention contains at least the peptide-bonded liposome of the present invention, and further contains other components as necessary.
  • the cytotoxic T lymphocyte activator and the peptide-bound liposome in the anti-tumor vaccine are the above-described peptide-bound liposomes of the present invention.
  • the content of the peptide-bound liposome in the cytotoxic T lymphocyte activator and the anti-tumor vaccine is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the peptide content is preferably 0.1% by mass to 100% by mass, more preferably 1% by mass to 99% by mass, and particularly preferably 10% by mass to 90% by mass.
  • the cytotoxic T lymphocyte activator and the anti-tumor vaccine may be composed of the peptide-bound liposome.
  • the cytotoxic T lymphocyte activator and other components in the anti-tumor vaccine are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. It is preferable to contain. There is no restriction
  • the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include aluminum hydroxide gel, complete Freund's adjuvant, incomplete Freund's adjuvant, pertussis adjuvant, poly (I, C), CpG- DNA etc. are mentioned. These may be used individually by 1 type and may use 2 or more types together.
  • CpG-DNA is preferable.
  • the CpG-DNA is a DNA containing a bacterial unmethylated CpG motif and is known to act as a ligand for a specific receptor (Toll-like receptor 9) (Biochim. Biophys. Acta 1489, 107- 116 (1999), Curr. Opin. Microbiol. 6, 472-477 (2003)).
  • the CpG-DNA can enhance the induction of cytotoxic T lymphocytes by the peptide-bound liposomes by activating dendritic cells (DC).
  • DC dendritic cells
  • the content of the adjuvant in the cytotoxic T lymphocyte activator and the antitumor vaccine is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.01% by mass to 10% by mass. % Is preferable, and 0.1% by mass to 5% by mass is more preferable.
  • the cytotoxic T lymphocyte activator and the anti-tumor vaccine may be administered as the peptide-bound liposome itself, which is an active ingredient thereof, or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition may comprise the peptide-bound liposome and a pharmacologically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • the cytotoxic T lymphocyte activator and the anti-tumor vaccine may be used in a mode combined with a pharmaceutical composition containing other ingredients as active ingredients.
  • the cytotoxic T lymphocyte activator and the antitumor vaccine may be used in a state of being blended in a medicament containing other ingredients as active ingredients.
  • the cytotoxic T lymphocyte activator and the anti-tumor vaccine can be formulated according to a conventional method.
  • the peptide-bonded liposome has low toxicity and can be administered as it is as a solution or as a pharmaceutical composition in an appropriate dosage form, orally or parenterally (for example, intravascular administration, subcutaneous administration, etc.) It is preferably administered parenterally.
  • the pharmaceutical composition for parenteral administration is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include injections and suppositories.
  • Examples of the injection include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection.
  • the injection can be prepared according to a known method.
  • Examples of the preparation method of the injection include dissolving or suspending the peptide-bonded liposome in a sterile aqueous solvent usually used for injection.
  • the aqueous solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, and the like. Examples include a buffer solution.
  • the pH of the aqueous solvent is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 to 10, and more preferably 6 to 8.
  • the prepared injection solution is preferably filled in a suitable ampoule.
  • a powder formulation of the peptide-bound liposome can be prepared by subjecting the suspension of the peptide-bound liposome to a treatment such as vacuum drying or freeze-drying.
  • the peptide-bonded liposomes can be stored in a powder state, and can be used by dispersing the powder with an aqueous solvent for injection at the time of use.
  • the dosage, administration timing, administration interval, and administration target of the cytotoxic T lymphocyte activator and the anti-tumor vaccine are not particularly limited and can be appropriately selected depending on the purpose.
  • the dosage can be appropriately selected in consideration of the subject to be administered, the administration method, the dosage form, and the like.
  • it when activating cytotoxic T lymphocytes in vivo by subcutaneous administration or nasal administration, it is usually 1 ⁇ g per peptide as a peptide in the peptide-bound liposome per adult (body weight 60 kg). ⁇ 1,000 ⁇ g is preferable, and 20 ⁇ g to 100 ⁇ g is more preferable.
  • 1 ⁇ g to 1,000 ⁇ g is preferable, preferably 20 ⁇ g to 100 ⁇ g per peptide as the peptide in the peptide-bound liposome per adult (body weight 60 kg). Is more preferable.
  • the administration timing and the administration interval are from twice over 4 weeks to 18 months. For example, it may be administered three times. In addition, for example, when a tumor is prevented or treated by subcutaneous administration, it may be administered 2 to 3 times for 4 to 18 months.
  • a human, a non-human mammal, birds etc. are mentioned.
  • a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, a mouse etc. are mentioned.
  • a chicken, a goose, a duck, an ostrich, a quail etc. are mentioned.
  • humans and mice are preferable, humans expressing HLA-A2, humans expressing HLA-A24 are more preferable, and humans expressing HLA-A2 are particularly preferable.
  • the anti-tumor vaccine is a vaccine targeting TERT, which is a pan-tumor antigen, it is considered that it can be applied to various tumors.
  • the peptide-bound liposome of the present invention can strongly induce cytotoxic T lymphocyte immunity that cross-reacts with an endogenous low affinity antigen (SEQ ID NO: 13). Therefore, it is considered that an excellent antitumor effect can be obtained even for cancer-bearing patients for whom an antitumor vaccine using a conventional high affinity antigen is ineffective.
  • the present invention is a method for preventing or treating a tumor, which comprises administering to the individual at least one of the cytotoxic T lymphocyte activator and the anti-tumor vaccine. It also relates to a method for preventing or treating tumors.
  • Test Example 1 Selection of candidate epitope
  • Test Example 1-1 Selection of natural epitope> About the amino acid sequence of human TERT (GenBank accession number: NP_001180305) and the amino acid sequence of mouse TERT (GenBank accession number: NP_03380), BIMAS (Bioinformatics and Molecular Analysis and Computational Biosciences, Computational Biosciences, Computational Biosciences, Computational Biosciences, Computational Biosciences of Computational Bioscience, Center for Information Technology, National Institutes of Health) using HLA-A binding to HLA-A2, one of the human MHC class I Analyzing the 2-binding peptide motif and predicting a 9-amino acid HLA-A2-restricted CTL epitope, an epitope having an amino acid sequence common to human TERT and mouse TERT.
  • Ten candidate epitopes (hereinafter sometimes referred to as “natural epitopes”) were selected in descending order of BIMAS score.
  • # 1 -Natural epitope- ILAKFLHWL
  • # 1 corresponds to positions 540 to 548 in the human TERT sequence.
  • RLFFYRKSV SEQ ID NO: 3, hereinafter sometimes referred to as “# 3”
  • # 3 corresponds to the 572nd to 580th positions in the human TERT sequence.
  • LLQAYRFHA SEQ ID NO: 5, hereinafter sometimes referred to as “# 5”
  • # 5 corresponds to positions 943 to 951 in the human TERT sequence.
  • LQVNSLQTV (SEQ ID NO: 7, hereinafter sometimes referred to as “# 7”)
  • the # 7 corresponds to the 926th to 934th positions in the human TERT sequence.
  • SVWSKLQSI (SEQ ID NO: 9, hereinafter sometimes referred to as “# 9”)
  • the # 9 corresponds to the 579th to 587th positions in the human TERT sequence.
  • QAYRFHACV (SEQ ID NO: 11, hereinafter sometimes referred to as “# 11”)
  • the # 11 corresponds to positions 945 to 953 in the human TERT sequence.
  • LQAYRFHAC (SEQ ID NO: 13, hereinafter sometimes referred to as “# 13”)
  • the # 13 corresponds to positions 944 to 952 in the human TERT sequence.
  • PLATFVRRL (SEQ ID NO: 15, hereinafter sometimes referred to as “# 15”)
  • the # 15 corresponds to the 23rd to 31st positions in the human TERT sequence.
  • GDMEMKLFA (SEQ ID NO: 17, hereinafter sometimes referred to as “# 17”)
  • the # 17 corresponds to the 847th to 855th positions in the human TERT sequence.
  • DLQVNSLQT (SEQ ID NO: 19, hereinafter sometimes referred to as “# 19”)
  • the # 19 corresponds to positions 925 to 933 in the human TERT sequence.
  • modified epitope Modified amino acids obtained by performing the following three types of amino acid substitutions on the natural epitopes of # 1, # 3, # 5, # 7, # 9, # 11, # 13, # 15, # 17, and # 19 Sequences were generated and affinity for HLA-A2 was predicted by BIMAS. Among the modified amino acid sequences in the respective natural epitopes, those having the highest BIMAS score were selected as candidate epitopes (hereinafter sometimes referred to as “modified epitopes”).
  • modified epitopes those having the highest BIMAS score were selected as candidate epitopes (hereinafter sometimes referred to as “modified epitopes”).
  • modified epitopes those having the highest BIMAS score were selected as candidate epitopes (hereinafter sometimes referred to as “modified epitopes”).
  • modified epitopes those having the highest BIMAS score were selected as candidate epitopes (hereinafter sometimes referred to as “modified epitopes”).
  • Y tyrosine
  • L leucine
  • SLWSKLQSI (SEQ ID NO: 10, hereinafter sometimes referred to as “# 10”)
  • # 10 the second amino acid in # 9 is replaced with L.
  • QLYRFHACV (SEQ ID NO: 12, hereinafter sometimes referred to as “# 12”)
  • # 12 the second amino acid in # 11 is replaced with L.
  • LQAYRFHAV (SEQ ID NO: 14, hereinafter sometimes referred to as “# 14”)
  • the ninth amino acid in # 13 is replaced with V.
  • YLATFVRRL (SEQ ID NO: 16, hereinafter sometimes referred to as “# 16”)
  • # 16 the first amino acid in # 15 is replaced with Y.
  • GLMENKLFA SEQ ID NO: 18, hereinafter sometimes referred to as “# 18”
  • # 18 the second amino acid in # 17 is replaced with L.
  • YLQVNSLQT SEQ ID NO: 20, hereinafter sometimes referred to as “# 20”
  • # 20 the first amino acid in # 19 is replaced with Y.
  • Table 1 shows the BIMAS scores of the natural epitope and the modified epitope.
  • Test production example 1 Production of peptide
  • the peptides of natural epitopes # 1 to # 20 selected in Test Example 1 and modified epitope peptides were artificially synthesized (manufactured by Eurofin Genomics).
  • lipid mixed powder Dioleoylphosphatidylcholine 1.3354 g (1.6987 mmol), the succinimide group-dioleoylphosphatidylethanolamine 0.2886 g (0.2831 mmol), cholesterol 0.7663 g (1.9818 mmol), and sodium dioleoylphosphatidylglycerol 0.4513 g (0.5662 mmol) of salt was placed in an eggplant-shaped flask, and 50 mL of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) was added and dissolved at 40 ° C.
  • the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Furthermore, 30 mL of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen and dried in a freeze dryer for 24 hours to obtain a lipid mixed powder.
  • This reaction solution was subjected to gel filtration according to a conventional method using Sepharose CL-4B equilibrated with a buffer solution. Since the liposome fraction is cloudy, the target fraction can be easily confirmed, but it may be confirmed with a UV detector or the like. Measure the phosphorous concentration in the resulting liposome suspension (measured using Phospholipid C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.)), and adjust the concentration so that the phospholipid-derived phosphorus concentration is 2 mM. The dilution was adjusted with a buffer solution to obtain four types of peptide-bound liposome suspensions.
  • H-2D which is a mouse-specific MHC class I gene and mouse ⁇ 2-microglobulin gene are knocked out, human HLA-A2 and human ⁇ 2-microglobulin gene are introduced, and expressed HHD mice (Pascolo S, Bervas N, Ure JM, Smith AG, Lemonier FA, Pernarau B. The Journal of Experimental Medicine 1997; 185 (12): 2043-2051).
  • booster immunization was performed in the same manner as the initial immunization.
  • lymphocytes in the mouse spleen were isolated.
  • the lymphocytes were antigen-stimulated with the corresponding epitope peptide (10 ⁇ g / mL) for 5 hours in the presence of FITC-labeled anti-CD107a antibody (manufactured by BD).
  • the antigen-stimulated cells are collected, the cell surface is stained with PerCP-labeled anti-CD8 antibody (manufactured by Biolegend), the cells are fixed, the cell membrane is permeabilized, and the inside of the cells is then labeled with PE-labeled anti-IFN- ⁇ .
  • CD107a-positive and IFN- ⁇ -positive lymphocytes in CD8-positive lymphocytes (hereinafter referred to as “CD107a + IFN- ⁇ + CD8) stained with an antibody (manufactured by Biolegend), specifically reacted with each epitope peptide and activated.
  • the percentage of “ + lymphocytes” may be measured by flow cytometry. The results are shown in FIGS. 1A to 1T and Table 2.
  • FIGS. 1A to 1T show the results when epitope peptides # 1 to # 20 were used, respectively. Induction of anti-tumor CTL in # 7, # 11, # 13, # 2, # 6, # 8, # 10, # 14, # 16, and # 18 from the results of FIGS. 1A to 1T and Table 2 was confirmed. Among these, the modified epitopes # 8, # 10, and # 14 had a high proportion of CD107a + IFN- ⁇ + CD8 + lymphocytes.
  • Test Example 3 Selection of immunogenic epitope peptide-2) ⁇ Production of peptide-bound liposome> A peptide-bonded liposome was prepared in the same manner as in Test Example 2 except that the modified peptide peptide # 8, # 10, or # 14 was used alone except that the pooled peptide was used in Test Example 2. did.
  • booster immunization was performed in the same manner as the initial immunization.
  • lymphocytes in the mouse spleen were isolated.
  • the lymphocytes were either (1) the corresponding epitope peptide (10 ⁇ g / mL), or (2) the natural epitope peptide of the corresponding epitope peptide (the corresponding epitope peptide was The natural epitope peptide in the case of # 8 is # 7, the natural epitope peptide in the case of # 10 is # 9, the natural epitope peptide in the case of # 14 is # 13) for 5 hours, Antigen stimulated.
  • the antigen-stimulated cells are collected, the cell surface is stained with PerCP-labeled anti-CD8 antibody (manufactured by Biolegend), the cells are fixed, the cell membrane is permeabilized, and the inside of the cells is then labeled with PE-labeled anti-IFN- ⁇ .
  • the percentage of CD107a + IFN- ⁇ + CD8 + lymphocytes that were stained with an antibody (manufactured by Biolegend) and specifically reacted and activated with each epitope peptide was measured by flow cytometry. The results are shown in FIGS. 2A to 2B.
  • FIG. 2A shows the results of performing the antigen stimulation with the corresponding epitope peptide
  • FIG. 2B shows the results of performing the antigen stimulation with the natural epitope peptide of the corresponding epitope peptide. From the results of FIGS. 2A to 2B, cross-reaction with the natural epitope peptide was observed for all the modified epitope peptides. In particular, a strong reaction was observed at # 14. The immune response induced by the peptide-bonded liposomes using # 14 was very strong compared to the immune response caused by # 13, the natural sequence of # 14.
  • Test Example 4 Selection of epitope peptide having immunogenicity-3) ⁇ Production of peptide-bound liposome> Peptide-bound liposomes were prepared in the same manner as in Test Example 3 except that the peptide was changed to # 1, # 13, or # 14.
  • the # 1 is a TERT dominant epitope and has the highest BIMAS score, and was used as a control.
  • booster immunization was performed in the same manner as the initial immunization.
  • lymphocytes in the mouse spleen were isolated.
  • the lymphocytes were treated with (1) the epitope peptide used for the immunization or (2) the natural epitope peptide of the epitope peptide used for the immunization for 5 hours. I was stimulated.
  • the antigen-stimulated cells are collected, the cell surface is stained with PerCP-labeled anti-CD8 antibody (manufactured by Biolegend), the cells are fixed, the cell membrane is permeabilized, and the inside of the cells is then labeled with PE-labeled anti-IFN- ⁇ .
  • the percentage of CD107a + IFN- ⁇ + CD8 + lymphocytes that were stained with an antibody (manufactured by Biolegend) and specifically reacted and activated with each epitope peptide was measured by flow cytometry. The results are shown in FIG.
  • mice immunized with peptide-bound liposomes using # 14 as the epitope peptide the proportion of CD107a + IFN- ⁇ + CD8 + lymphocytes was immunized with peptide-bound liposomes using # 1 as the epitope peptide. It was very high compared to the mouse.
  • booster immunization was performed in the same manner as the initial immunization.
  • lymphocytes in the mouse spleen were isolated.
  • the separated lymphocytes were cultured for 6 days in the presence of the epitope peptide used for the immunization.
  • the lymphocytes were stimulated with TERT-positive HLA-A2-positive mouse tumor cells RMAHHD (obtained from Dr. Lemmonier, Pasteur Institute, France) for 5 hours.
  • RMAHHD obtained from Dr. Lemmonier, Pasteur Institute, France
  • the cell surface is stained with PerCP-labeled anti-CD8 antibody (manufactured by Biolegend), the cells are fixed, the cell membrane is permeabilized, and the cell interior is stained with PE-labeled anti-IFN- ⁇ antibody (manufactured by Biolegend).
  • IFN- ⁇ + CD8 + lymphocytes The proportion of IFN- ⁇ -positive lymphocytes (hereinafter sometimes referred to as “IFN- ⁇ + CD8 + lymphocytes”) in CD8-positive lymphocytes that were activated in response to tumor cells was measured by flow cytometry. The results are shown in FIG.
  • control epitope is an epitope derived from a hepatitis C virus core protein that is unrelated to a tumor transplanted to a mouse described later. Peptide and used as a control.
  • mice were used. In addition, 5 to 6 mice were used per group.
  • booster immunization was performed in the same manner as the initial immunization.
  • TERT positive HLA-A2 positive mouse tumor cells RMAHHD obtained from Dr. Lemmonner, Pasteur Institute, France
  • the area of the tumor was measured with a caliper every 3 to 4 days to examine the growth of the tumor tissue.
  • the results of measuring the area of the tumor 24 days after the transplantation are shown in FIG.
  • the mouse was euthanized. .
  • mice immunized with peptide-bound liposomes conjugated with control epitopes all mice were euthanized after 24 days of transplantation with tumors exceeding 1,000 mm 2 in tumor area.
  • mice immunized with peptide-bound liposomes conjugated with # 13 the tumor area was small compared to mice immunized with peptide-bound liposomes conjugated with the control epitope at the 24th day after transplantation, No statistically significant difference was obtained.
  • mice immunized with the peptide-bonded liposomes to which # 14 was bound the tumor area was clearly compared with the mice immunized with the peptide-bound liposomes to which the control epitope was bound at the 24th day after the transplantation. Small and statistically significant differences were observed (Man-Whitney U test, p ⁇ 0.01).
  • the # 14 is a modification of # 13, which is a novel low immunogenic human TERT-derived antigen that has not been reported so far, and is considered to be a low immunogenic tumor antigen.
  • the peptide-bonded liposome to which # 14 was bound had an antitumor immunity inducing ability, a tumor growth suppressing effect, and a survival time extending effect.
  • the antitumor vaccine of the present invention containing the peptide-bonded liposome of the present invention is superior in terms of immunity induction effect and quality stability, compared to peptide vaccines and cell vaccines that are the mainstream of current antitumor vaccines. ing.
  • the anti-tumor vaccine of the present invention enables appropriate antigen presentation to T cells and can induce a cellular immune response to low immunogenic tumor antigens, avoiding immune tolerance to tumor antigens
  • it is an anti-tumor vaccine preparation having an unprecedented feature that can provide an anti-tumor effect even for cancer-bearing patients for whom an anti-tumor vaccine using a high affinity antigen is ineffective, and can be an excellent vaccine for tumor treatment I can expect.
  • the anti-tumor vaccine of the present invention is a vaccine targeting TERT, which is a pan-tumor antigen, and is considered to be applicable to various neoplastic diseases.
  • Examples of the aspect of the present invention include the following.
  • ⁇ 1> A peptide-bonded liposome characterized in that a peptide comprising the amino acid sequence represented by SEQ ID NO: 14 is bound to the surface of the liposome.
  • ⁇ 2> A phospholipid having any one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond; It is a peptide bond liposome as described in said ⁇ 1> containing a stabilizer.
  • ⁇ 3> The peptide-bonded liposome according to ⁇ 2>, wherein the phospholipid is a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond.
  • ⁇ 4> The peptide-bonded liposome according to any one of ⁇ 2> to ⁇ 3>, wherein the acyl group is an oleyl group.
  • the aforementioned phospholipid is at least one selected from diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine
  • ⁇ 6> The peptide-bonded liposome according to any one of ⁇ 2> to ⁇ 5>, wherein the liposome stabilizer is cholesterol.
  • the peptide binds to a phospholipid having any one of an acyl group having 14 to 24 carbon atoms having one unsaturated bond and a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond.
  • ⁇ 8> A cytotoxic T lymphocyte activator comprising the peptide-bonded liposome according to any one of ⁇ 1> to ⁇ 7>.
  • ⁇ 9> The cytotoxic T lymphocyte activator according to ⁇ 8>, further comprising an adjuvant.
  • An antitumor vaccine comprising the peptide-bonded liposome according to any one of ⁇ 1> to ⁇ 7>.
  • ⁇ 12> A method for preventing or treating a tumor, wherein the cytotoxic T lymphocyte activator according to any one of ⁇ 8> to ⁇ 9>, and ⁇ 10> to ⁇ 10>11>.
  • a method comprising administering at least one of the antitumor vaccines according to any one of 11).
  • ⁇ 14> Use of the peptide-bonded liposome according to any one of ⁇ 1> to ⁇ 7> for producing a cytotoxic T lymphocyte activator.
  • ⁇ 15> Use of the peptide-bonded liposome according to any one of ⁇ 1> to ⁇ 7> for producing an antitumor vaccine.

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Abstract

L'invention concerne un liposome à liaison peptidique dans lequel un peptide constitué d'une séquence d'acides aminés représentée par la SEQ ID n°14, est lié à la surface d'un liposome.
PCT/JP2015/076214 2014-09-26 2015-09-16 Liposome à liaison peptidique, activateur de lymphocyte t cytotoxique, et vaccin antitumoral Ceased WO2016047509A1 (fr)

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JP2014005205A (ja) * 2010-10-14 2014-01-16 Saitama Medical Univ エボラウイルスリポソームワクチン

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