HK1215440B - Novel peptide having 5 linked ctl epitopes - Google Patents

Description

Novel peptides having 5 linked CTL epitopes

Technical Field

The present invention relates to a novel peptide useful as a cancer antigen peptide. 3 more 3 particularly 3, 3 the 3 present 3 invention 3 relates 3 to 3 a 3 novel 3 cancer 3 antigen 3 peptide 3 in 3 which 35 3 peptides 3 having 3 HLA 3- 3 a 3- 3 restricted 3ctl 3 inducibility 3 are 3 linked 3, 3 and 3 a 3 pharmaceutical 3 composition 3 using 3 the 3 same 3. 3

Background

Cancer accounts for the 1 st cause of death in Japanese, about 35 million people die a year, and is still a serious disease at present. The established cancer therapies include surgical resection, anticancer drug therapy, and radiotherapy. However, the therapy has problems such as recurrence, reduction in quality of life (QOL), and no other treatment options in the case of intermediate-stage cancer that cannot receive the aforementioned therapy.

Cancer immunotherapy (cancer vaccine therapy) has long been expected as a new therapy, and clinical studies of cancer peptide vaccines have been started worldwide since 1990 when epitope peptides in human tumor antigens can be identified. However, as a result of analysis of results of clinical studies in the treatment with the peptide alone or in combination, it was found that the success rate was only 2.7% in 1,000 or more administration examples (non-patent document 1), and it was difficult to put it into practical use.

On the other hand, clinical studies using a specific cancer peptide vaccine for a long period of time are continued in japan, and the results thereof are gradually becoming remarkable. In recent years, as one of the methods aimed at improving the therapeutic performance, a strategy of administering a plurality of cancer peptides instead of administering 1 cancer peptide has also been adopted. For example, a cancer peptide vaccine therapy of a tailor-made (tailor-made) type, in which a plurality of peptides to be administered are selected, is performed by examining the HLA type and specific immune response of a patient in advance, and safety and antitumor effect are confirmed. Specifically, by administering a tailored peptide vaccine alone or in combination with an anticancer agent, excellent clinical effects and safety are achieved in brain tumors, cervical cancer, prostate cancer, and pancreatic cancer (non-patent documents 2, 3, and 4).

In cancer peptide vaccine therapy, the cellular immunity by epitope-specific cytotoxic T lymphocytes (hereinafter abbreviated as CTLs), which are considered to be main effector cells, is HLA-restricted, and cancer peptide vaccine development has been carried out for patients having a specific HLA classification, specifically, only patients having HLA-a2 or HLA-a24 classification in large numbers of target patients.

However, these 2 kinds of HLA types hold about 40% and 60% in japan (non-patent document 5), respectively, and there is a problem that patients with HLA types other than these 2 kinds cannot receive the benefit of cancer peptide vaccines. Therefore, the need for HLA typing examination before treatment starts is one cause of delay in the start timing of treatment and increase in the burden on the patient. From the above, development and research of a cancer peptide vaccine applicable to all cancer patients without requiring HLA typing test are desired.

In addition, it is known that in cancer peptide vaccine therapy, in addition to activation of CTLs as cellular immunity, the induction of immunoglobulin production as humoral immunity contributes to the effect of prolonging life (non-patent document 6).

Documents of the prior art

Non-patent document

Non-patent document 1: rosenberg SA et al, Nature Med.2004,10(9):909-15

Non-patent document 2: terasaki M et al, J Clin Oncol.2011,29(3):337-44

Non-patent document 3: noguchi M et al, Cancer lmmunol.lmmunother.2010,59(7):1001-9

Non-patent document 4: yanagimoto H et al, Cancer Sci.2007,98(4):605-11

Non-patent document 5: set A et al, immunogenetics.1999,50(3-4):201-12

Non-patent document 6: noguchi M et al, Cancer biol. Ther.2011,10(12):1266-79

Disclosure of Invention

An object of the present invention is to provide a cancer antigen peptide which can be administered as a cancer peptide vaccine to a wide range of cancer patients without requiring HLA typing examination and is not limited to patients having a specific HLA typing.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that: 3 A 3 peptide 3 having 35 3 linked 3CTL 3 epitopes 3 obtained 3 by 3 linking 35 3 peptides 3 selected 3 from 3CTL 3 epitope 3 peptides 3 reported 3 to 3 have 3 the 3 induction 3 ability 3 of 3 any 3 one 3 of 3 HLA 3- 3 A 32 3, 3 HLA 3- 3 A 3 24 3, 3 HLA 3- 3 A 3 26 3 and 3 HLA 3- 3 A 33 3 supertypes 3 or 3 the 3 induction 3 ability 3 of 3 the 3 above 3- 3 mentioned 3 various 3 HLA 3- 3 A 3 restricted 3 CTLs 3 via 3 a 3 linker 3 can 3 be 3 administered 3 to 3 a 3 wide 3 range 3 of 3 cancer 3 patients 3 without 3 requiring 3 HLA 3 typing 3 examination 3 and 3 without 3 being 3 limited 3 to 3 patients 3 having 3 a 3 specific 3 HLA 3 type 3, 3 thereby 3 strongly 3 inducing 3 CTLs 3 and 3 immunoglobulins 3 specific 3 to 3 the 3CTL 3 epitope 3 peptides 3 constituting 3 the 3 peptide 3, 3 and 3 the 3 present 3 invention 3 has 3 been 3 completed 3. 3

That is, the present invention has the following features.

[1] A peptide having 5 linked epitopes selected from the group consisting of CTL epitope peptides consisting of: a peptide represented by the sequence No. 1 (PEP1), a peptide represented by the sequence No. 2 (PEP2), a peptide represented by the sequence No. 3 (PEP3), a peptide represented by the sequence No. 4 (PEP4), a peptide represented by the sequence No. 5 (PEP5), a peptide represented by the sequence No. 6 (PEP6), and a peptide represented by the sequence No. 7 (PEP7), wherein 5 peptides which can be repeatedly selected from the group consisting of the peptides represented by the sequence No. 1 (PEP1), the peptides represented by the sequence No. 2 (PEP 35

The peptide having 5 linked epitopes has one or more characteristics selected from the following (1) to (12):

(1) a sequence comprising PEP5 and PEP2 adjacent to each other in the order from the N-terminal side via a linker;

(2) a sequence comprising PEP2 and PEP4 adjacent to each other in the order from the N-terminal side via a linker;

(3) a sequence comprising PEP4 and PEP6 adjacent to each other in the order from the N-terminal side via a linker;

(4) a sequence comprising PEP6 and PEP3 adjacent to each other in the order from the N-terminal side via a linker;

(5) a sequence comprising PEP4 and PEP1 adjacent to each other in the order from the N-terminal side via a linker;

(6) a sequence comprising PEP1 and PEP3 adjacent to each other in the order from the N-terminal side via a linker;

(7) a sequence comprising PEP4 and PEP2 adjacent to each other in the order from the N-terminal side via a linker;

(8) a sequence comprising PEP1 and PEP4 adjacent to each other in the order from the N-terminal side via a linker;

(9) a sequence comprising PEP5 and PEP1 adjacent to each other in the order from the N-terminal side via a linker;

(10) a sequence comprising PEP6 and PEP5 adjacent to each other in the order from the N-terminal side via a linker;

(11) contains PEP2 at the C-terminal end; and

(12) contains PEP3 at the C-terminus.

[2] The peptide having 5 linked epitopes according to [1], which comprises a sequence containing PEP5 and PEP2, PEP6 and PEP5, or PEP4 and PEP6 adjacent in order from the N-terminal side via a linker at the N-terminal and/or a sequence containing PEP7 and PEP3, PEP4 and PEP3, PEP6 and PEP3, PEP1 and PEP3, PEP5 and PEP2, PEP4 and PEP2, or PEP5 and PEP3 adjacent in order from the N-terminal side via a linker at the C-terminal.

[3] The peptide having 5 linked epitopes according to [2], which comprises a sequence selected from the following (a) to (p):

(a)PEP5-PEP2-PEP4-PEP7-PEP3；

(b)PEP5-PEP2-PEP7-PEP3-PEP4；

(c)PEP4-PEP6-PEP5-PEP7-PEP3；

(d)PEP5-PEP2-PEP4-PEP6-PEP3；

(e)PEP5-PEP2-PEP4-PEP1-PEP3；

(f)PEP4-PEP5-PEP2-PEP7-PEP3；

(g)PEP7-PEP3-PEP4-PEP5-PEP2；

(h)PEP5-PEP7-PEP3-PEP4-PEP2；

(i)PEP6-PEP1-PEP4-PEP5-PEP2；

(j)PEP6-PEP5-PEP1-PEP4-PEP2；

(k)PEP4-PEP5-PEP1-PEP6-PEP3；

(l)PEP7-PEP2-PEP4-PEP5-PEP3；

(m)PEP4-PEP2-PEP7-PEP5-PEP3；

(n)PEP7-PEP2-PEP5-PEP4-PEP3；

(o) PEP5-PEP2-PEP7-PEP4-PEP 3; or

(p)PEP5-PEP2-PEP4-PEP3-PEP7；

Wherein "-" in the formula represents a linker.

[4] The peptide having 5 linked epitopes according to any one of [1] to [3], wherein the linker is an amino acid linker.

[5] The peptide having 5 linked epitopes according to [4], wherein the amino acid linker is an arginine dimer linking 2 arginines.

[6] A CTL obtained by stimulating peripheral blood lymphocytes with the peptide having 5 linked epitopes according to any one of [1] to [5 ].

[7] A pharmaceutical composition for treating or preventing cancer, which comprises the peptide having 5 linked epitopes according to any one of [1] to [5], or the CTL according to [6] as an active ingredient.

[8] The pharmaceutical composition according to [7], which is an immunotherapeutic agent.

[9] A method of treating cancer, comprising: a peptide having 5 linked epitopes according to any one of [1] to [5], or a CTL according to [6] is administered to a cancer patient.

[10] A method of immunizing a subject against cancer, comprising: a peptide having 5 linked epitopes according to any one of [1] to [5], or a CTL according to [6] is administered to a subject.

The present specification includes the contents described in the specification and/or drawings of japanese patent application No. 2013-047271, which is the priority basis of the present application.

Effects of the invention

According to the present invention, it is possible to provide a cancer antigen peptide which can be administered as a cancer peptide vaccine to a wide range of cancer patients without requiring HLA typing examination and is not limited to patients having a specific HLA type.

The peptide having 5 linked CTL epitopes of the present invention can be administered to a wide range of cancer patients, for example, HLA-a 2-positive patient population, HLA-a 24-positive patient population, HLA-a 26-positive patient population, HLA-A3 supertype-positive patient population, without requiring HLA typing examination, and can treat and/or prevent cancer in the patient or a disease resulting therefrom. In addition, since it is observed that the tumor antigen constituting the peptide having 5 linked CTL epitopes of the present invention is expressed in a plurality of cancer species, the peptide having 5 linked CTL epitopes of the present invention can be used as a pharmaceutical composition (more specifically, an immunotherapeutic agent) for treating or preventing various cancer species. Furthermore, by administering the peptide having 5 linked epitopes of the present invention, CTL and immunoglobulin specific to CTL epitope peptides can be more strongly induced and antitumor immunity can be more effectively activated, as compared with the case where the constituted CTL epitope peptides are mixed and administered in equal amounts.

Drawings

FIG. 1 shows the results of epitope-specific CTL induction in a mouse model administered with a peptide having 5 linked epitopes (shown as not studied).

FIG. 2 shows the results of the determination of epitope-specific IgG antibody titers in serum in a mouse model administered with a peptide having 5 linked epitopes.

Detailed Description

The present invention will be further specifically described below.

1. Peptides having 5 linked epitopes

The "peptide having 5 linked epitopes" of the present invention means: 5 peptides selected from among CTL epitope peptides derived from the same and/or different tumor antigen molecules are linearly linked via a linker and set as 1 molecule of peptide.

"CTL epitope peptide derived from tumor antigen molecule" is a peptide produced by tumor antigen decomposition in tumor cells, which can be recognized by tumor-specific CTLs, and/or can induce and/or activate tumor-specific CTLs by binding to HLA class I molecules and presenting on the cell surface. By "inducing tumor-specific CTLs" is meant: CTLs that specifically recognize CTL epitope peptides derived from tumor antigen molecules are allowed to differentiate and/or proliferate in vitro or in vivo. In addition, "activating tumor-specific CTLs" means: interferon gamma (IFN- γ) is produced by CTL recognizing antigens presented by HLA class I molecules. In the present specification, "CTL epitope peptide derived from tumor antigen molecules" is sometimes simply referred to as "CTL epitope peptide".

As the CTL epitope peptide used in the present invention, known CTL epitope peptides can be used, and the following CTL epitope peptides can be mentioned:

PEP 1: KLVERLGAA (SEQ ID NO: 1[ WO 0l/Ol1044 ]);

PEP 2: ASLDSDPWV (SEQ ID NO. 2[ WO 02/010369 ]);

PEP 3: LLQAEAPRL (SEQ ID NO: 3[ WO 00/12701 ]);

PEP 4: DYSARWNEI (SEQ ID NO. 4[ Japanese patent application laid-open No. 11-318455 ]);

PEP 5: VYDYNCHVDL (SEQ ID NO: 5[ WO 00/12701 ]);

PEP 6: LYAWEPSFL (SEQ ID NO: 6[ Japanese patent application laid-open No. 2003-000270 ]);

PEP 7: QIRPIFSNR (SEQ ID NO. 7[ Cancer lmmunol.lmmunother.2007, 56(5),689-698 ]).

In the present specification, the peptide represented by the sequence number 1 is sometimes referred to as "PEP 1", the peptide represented by the sequence number 2 is sometimes referred to as "PEP 2", the peptide represented by the sequence number 3 is sometimes referred to as "PEP 3", the peptide represented by the sequence number 4 is sometimes referred to as "PEP 4", the peptide represented by the sequence number 5 is sometimes referred to as "PEP 5", the peptide represented by the sequence number 6 is sometimes referred to as "PEP 6", and the peptide represented by the sequence number 7 is sometimes referred to as "PEP 7".

In the present invention, a peptide having an amino acid sequence having substitution, insertion, deletion, and/or addition of one or more amino acids in each of the amino acid sequences of PEP1, PEP2, PEP3, PEP4, PEP5, PEP6, and PEP7, and having CTL inducibility and immunoglobulin production inducibility equal to or higher than those of the original peptide, may be used as the "CTL epitope peptide". Here, the number of "plural" is 1 to 3, preferably 1 to 2. Examples of such peptides include peptides substituted with amino acids having similar properties to the original amino acids (i.e., peptides obtained by conservative amino acid substitution).

The PEP1, PEP2, PEP3, PEP4 and PEP5 are captured by HLA-A2, the PEP2, PEP4, PEP5 and PEP6 are captured by HLA-A24, the PEP2, PEP4, PEP5 and PEP7 are captured by HLA-A3 in a super-type manner, and the PEP2 and PEP5 are captured by HLA-A26, so that CTL can be induced and/or activated. In addition, the genes encoding these CTL epitope peptides were confirmed to be expressed in various Cancer species (Yang D et al, Cancer Res.1999,59:4056-63, Harashima N et al, Eur.J.Immunol.2001,31(2), 323-32).

5 peptides selected from PEP1, PEP2, PEP3, PEP4, PEP5, PEP6, and PEP7 (which may be selected repeatedly) are linearly linked via a linker, respectively.

The linker may be cleaved when a peptide having 5 linked epitopes is administered to an organism, and the linked CTL epitope peptides may be separated from each other, and examples thereof include: ester bonds, ether bonds, amide bonds, sugar chain linkers, polyethylene glycol linkers, amino acid linkers, and the like. Examples of the amino acid sequence used as the amino acid linker may include arginine dimer, lysine dimer, glycine pentamer, glycine hexamer, alanine-tyrosine (AAY), isoleucine-leucine-alanine (ILA), arginine-valine-lysine-arginine (RVKR), etc., preferably arginine dimer.

The selected CTL epitope peptides and their order of ligation can be determined by: 3 peptides 3 having 35 3 joined 3 epitopes 3 synthesized 3 in 3 the 3 given 3 combination 3 and 3 in 3 the 3 given 3 joining 3 order 3 were 3 administered 3 to 3 human 3 HLA 3- 3 A 3 transgenic 3 mice 3 and 3 evaluated 3 for 3 the 3 presence 3 or 3 absence 3 of 3CTL 3- 3 induction 3 specific 3 to 3 each 3CTL 3 epitope 3 peptide 3 in 3 the 3 organism 3. 3

Preferably, the peptide having 5 linked epitopes of the present invention has one or more characteristics selected from the following (1) to (12):

(1) a sequence comprising PEP5 and PEP2 adjacent to each other in the order from the N-terminal side via a linker;

(2) a sequence comprising PEP2 and PEP4 adjacent to each other in the order from the N-terminal side via a linker;

(3) a sequence comprising PEP4 and PEP6 adjacent to each other in the order from the N-terminal side via a linker;

(4) a sequence comprising PEP6 and PEP3 adjacent to each other in the order from the N-terminal side via a linker;

(5) a sequence comprising PEP4 and PEP1 adjacent to each other in the order from the N-terminal side via a linker;

(6) a sequence comprising PEP1 and PEP3 adjacent to each other in the order from the N-terminal side via a linker;

(7) a sequence comprising PEP4 and PEP2 adjacent to each other in the order from the N-terminal side via a linker;

(8) a sequence comprising PEP1 and PEP4 adjacent to each other in the order from the N-terminal side via a linker;

(9) a sequence comprising PEP5 and PEP1 adjacent to each other in the order from the N-terminal side via a linker;

(10) a sequence comprising PEP6 and PEP5 adjacent to each other in the order from the N-terminal side via a linker;

(11) contains PEP2 at the C-terminal end; and

(12) contains PEP3 at the C-terminus.

Preferably, the peptide of the invention having 5 linked epitopes comprises

(I) Containing at the N-terminus

PEP5 and PEP2,

PEP6 and PEP5, or

PEP4 and PEP6

Sequences which are adjacent in order from the N-terminal side via a linker, and/or

(II) containing at the C-terminus

PEP7 and PEP3,

PEP4 and PEP3,

PEP6 and PEP3,

PEP1 and PEP3,

PEP5 and PEP2,

PEP4 and PEP2, or

PEP5 and PEP3

Sequences adjacent in order from the N-terminal side via a linker.

Further preferably, the peptide having 5 linked epitopes of the present invention contains or consists of a sequence selected from the following (a) to (p) (wherein "-" represents a linker):

(a)PEP5-PEP2-PEP4-PEP7-PEP3；

(b)PEP5-PEP2-PEP7-PEP3-PEP4；

(c)PEP4-PEP6-PEP5-PEP7-PEP3；

(d)PEP5-PEP2-PEP4-PEP6-PEP3；

(e)PEP5-PEP2-PEP4-PEP1-PEP3；

(f)PEP4-PEP5-PEP2-PEP7-PEP3；

(g)PEP7-PEP3-PEP4-PEP5-PEP2；

(h)PEP5-PEP7-PEP3-PEP4-PEP2；

(i)PEP6-PEP1-PEP4-PEP5-PEP2；

(j)PEP6-PEP5-PEP1-PEP4-PEP2；

(k)PEP4-PEP5-PEP1-PEP6-PEP3；

(l)PEP7-PEP2-PEP4-PEP5-PEP3；

(m)PEP4-PEP2-PEP7-PEP5-PEP3；

(n)PEP7-PEP2-PEP5-PEP4-PEP3；

(o) PEP5-PEP2-PEP7-PEP4-PEP 3; or

(p)PEP5-PEP2-PEP4-PEP3-PEP7。

Among the peptides having 5 linked epitopes of the present invention, preferred 2, further preferred 3, more preferred 4, most preferred 5 CTL epitope peptides to the 5 CTL epitope peptides linked can induce and/or activate CTL specific to each CTL epitope peptide, and can induce immunoglobulin production specific to each CTL epitope peptide. Here, "immunoglobulin" refers to IgG, IgM, IgA, and IgD.

2. Production of peptides with 5 linked epitopes

the peptides of the present invention having 5 linked epitopes can be produced by conventional Peptide Synthesis Methods such as liquid Phase Synthesis Methods, Solid Phase Synthesis Methods, Peptide Synthesis Methods using automated Peptide synthesizers, etc. (Kelley et al, Genetics engineering principles and Methods, Setlow, J.K.eds., Plenum Press NY. (1990) Vol.12, p.1-19; Stewart et al, Solid-Phase Peptide Synthesis (1989) W.H.Freeman Co., Houghten, Proc.Natl.Acad.Sci.USA (1985)82: p.5132, < New chemistry Proc. seat 1 タンパク IV (1992) Japan Biochemical society coded, east chemico., Do.) for Peptide Synthesis, α -amino groups to be bound and the alpha-carboxy functional groups to be bound of each amino acid and selective removal of the protected amino groups from the Peptide-amino groups, the Peptide-protected amino groups are formed by successive Peptide-amino-Peptide-protecting groups, and the Peptide-protected amino-amino groups are removed from the protected amino groups, the Peptide-amino groups are generally, and the Peptide-protected amino-protected groups are removed by successive Peptide Synthesis procedures, and the Peptide-protected Peptide-amino group-protected Peptide-protected groups are generally described above.

Alternatively, the peptide can be produced by gene recombination method, phage display method, or the like using a nucleic acid encoding the peptide having 5 linked epitopes of the present invention.

The gene recombination method comprises the following steps: the DNA encoding the peptide having 5 linked epitopes of the present invention is inserted into an appropriate expression vector, the vector is introduced into an appropriate host cell, the cell is cultured, and the target peptide is recovered from the inside or from the extracellular fluid. The vector is not limited, and examples thereof include vectors such as plasmids, phages, cosmids, phagemids, and viruses. Host cells for introducing the vector include bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (e.g., mammalian cells), plant cells, etc., and transformation or transfection into these cells includes, for example, calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method, etc. The method of culturing the transformed cells is carried out according to the conventional method for culturing host organisms. In order to facilitate recovery of the peptide of the present invention, it is preferable that the peptide produced by expression is secreted extracellularly. For this purpose, DNA encoding a peptide sequence enabling secretion of the peptide from the cell is bound to the 5' -terminal side of the DNA encoding the target peptide. Alternatively, the target peptide accumulated in the cell may also be recovered. In this case, the target peptide is recovered by physically or chemically destroying the cells and using a protein purification technique.

The produced peptide can be recovered or purified by a conventional method, for example, by chromatography such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, HPLC, ammonium sulfate fractionation, ultrafiltration, immunoadsorption method and the like.

3. Cytotoxic T lymphocytes

Using the peptide having 5 linked epitopes of the present invention, CTL specific to CTL epitope peptides which damage cancer cells can be obtained in vitro. Methods for inducing CTLs in vitro using CTL epitope peptides are known (e.g., japanese patent application laid-open No. 2006-14637), and these methods can also be utilized in the present invention. For example, plate-adherent cells in Peripheral Blood Mononuclear Cells (PBMC) from healthy humans or cancer patients are cultured in the presence of cytokines such as GM-CSF, IL-4, etc., to induce Dendritic Cells (DCs). After the dendritic cells were shock-sensitized with the peptide having 5 linked epitopes of the present invention, X-ray irradiation was performed to prepare antigen presenting cells (stimulators). In the case where DC cannot be used, a substance subjected to X-ray irradiation after Peripheral Blood Mononuclear Cells (PBMCs) from a healthy person or the same cancer patient are pulsed with the peptide having 5 linked epitopes of the present invention. Next, Peripheral Blood Mononuclear Cells (PBMC) from healthy persons or Peripheral Blood Mononuclear Cells (PBMC) from cancer patients or lymphocytes (responder) in the relevant lymph nodes are added and cultured in the presence of cytokines such as IL-2, IL-4, IL-7, etc. Thereafter, the antigen-presenting cells obtained by pulse sensitization as described above were re-stimulated by adding the peptide having 5 linked epitopes of the present invention, and further cultured in the presence of cytokines such as IL-2 and the like.

for example, a medium in which various cytokines such as IL-2 and the like and Fetal Calf Serum (FCS) are added to RHAM α medium (LAKmedium described in Kawai, k., Sasaki, T., Saijo-Kurita, k., Akaza, h., Koiso, k., and Ohno, T., Cancer immunol.immunol.35, 225-229,1992), AIMV medium (GIBCO BRL, Life Technologies, INC.) or RPMI1640 medium, and the like can be used.

The culture conditions may be those known to those skilled in the art. For example, the culture temperature is set to 33 ℃ to 41 ℃, preferably 37 ℃. Alternatively, air or an appropriate concentration of oxygen and carbon dioxide (e.g., 5% CO) at an appropriate concentration to maintain the pH of the medium at about 7.4 may be included₂) The inert gas of (3) is used as a gas phase. Preferably, the culture is carried out for 4 to 10 days, more preferably for 7 days. CTLs induced by performing such culturing contain CTLs specific for each of the CTL epitope peptides of preferably 2, further preferably 3, more preferably 4, most preferably 5 CTL epitope peptides constituting the peptides having 5 linked epitopes, and thus they can specifically damage cancer cells.

4. Pharmaceutical composition

The peptide having 5 linked epitopes of the present invention can be utilized as an active ingredient of a pharmaceutical composition for use in immunotherapy of cancer.

In the pharmaceutical composition of the present invention, one or more of the above-mentioned peptides having 5 linked epitopes may be contained as an active ingredient. By containing a plurality of peptides having 5 linked epitopes, a stronger effect can be obtained.

It was observed that genes encoding PEP1, PEP2, PEP3, PEP4, PEP5, PEP6, and PEP7 contained in the peptide having 5 linked epitopes of the present invention were expressed in a plurality of solid cancers and hematological cancers. Examples of the solid cancer include: brain tumor, lung cancer, breast cancer, thyroid cancer, cervical cancer, uterine corpus cancer, ovarian cancer, esophageal cancer, stomach cancer, GIST, pancreatic cancer, colon cancer, rectal cancer, anal cancer, kidney cancer, liver cancer, biliary tract cancer, head and neck cancer, bladder cancer, prostate cancer, malignant melanoma, skin cancer, tongue cancer, osteosarcoma, chondrosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, and the like. Examples of hematological cancers include: leukemia, malignant lymphoma, myeloma, and the like. Therefore, the peptides of the present invention having 5 linked epitopes are useful for the treatment or prevention of these cancers. Here, "treatment or prevention of cancer" means prevention of occurrence or recurrence of cancer, suppression of progression or worsening of cancer, and amelioration of symptoms of cancer.

The pharmaceutical composition of the present invention may contain various conventional organic or inorganic carrier substances and the like as pharmaceutically acceptable preparation raw materials. Examples of the usable pharmaceutical carrier include a stabilizer, an antibacterial agent, a buffering agent, an isotonizing agent, a chelating agent, a pH adjuster, a surfactant (surfactant), a filler, a thickener, a binder, a wetting agent, a disintegrating agent, a surfactant (surfactant), a lubricant, a soothing agent, a diluent, an excipient, and the like, which are generally used depending on the application form of the preparation.

In addition, the pharmaceutical composition of the present invention may contain adjuvants known to be used in the administration of vaccines. As adjuvants, there may be mentioned: bacterial preparations such as Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), alum, lipid a, monophosphoryl lipid A, BCG (Bacillus-Calmette-gurrin), bacterial component preparations such as tuberculin, natural high molecular substances such as keyhole limpet hemocyanin and zymomannan, muramyl tripeptides or muramyl dipeptides or derivatives thereof, alum (alum), nonionic block copolymers, and the like, and 1 or a combination of 2 or more of these substances may be used. Adjuvants may be administered simultaneously with the pharmaceutical composition of the invention, either in admixture or as an emulsion.

Further, in addition to the above-described peptides having 5 linked epitopes, the pharmaceutical composition of the present invention may contain one or more combinations of the following: known CTL epitope peptides derived from tumor antigen molecules, peptides containing the peptides, or peptides obtained by linking the peptides (hereinafter, referred to as "known CTL epitope peptides derived from tumor antigen molecules, etc.). As well-known CTL epitope peptides derived from tumor antigen molecules, there can be mentioned, for example: WT-1 p126-134, modified (M236Y) WT-1 p235-243, NY-ESO-1p157-165, modified (T210M) gp100 p209-217, survivin-2B p80-88, Her-2/neu p63-71, VEGFR2p169-177, MART-1 p26-35, glypican-3 p298-306, SPARC p143-151, etc., but are not limited thereto. In this case, the peptide having 5 linked epitopes of the present invention and the known CTL epitope peptide derived from a tumor antigen molecule may be in the form of a single-dose preparation, or a preparation containing the peptide having 5 linked epitopes of the present invention as an active ingredient and a preparation containing the known CTL epitope peptide derived from a tumor antigen molecule as an active ingredient may be in different forms.

The pharmaceutical composition of the present invention may be formulated in a selected form depending on the administration form. Typical preparation forms include, but are not limited to, liquid preparations, emulsions, liposome preparations, lipid emulsions, inclusion compounds such as cyclodextrins, suspensions, ointments, creams, transdermal absorbents, transmucosal absorbents, tablets, pills, capsules, powders, granules, fine granules, and syrups. These preparations are further classified into oral agents, non-oral agents, nasal agents, vaginal agents, suppositories, sublingual agents, inhalants, eye drops, ear drops and the like according to the administration route, and these preparations can be prepared, shaped or prepared according to a conventional method, respectively. In addition to being used as a liquid preparation, these preparations may be used after being stored in a freeze-dried state and then dissolved with a buffer solution containing water, physiological saline, or the like to be prepared at an appropriate concentration at the time of use.

In addition, the pharmaceutical composition of the present invention may contain, as an active ingredient, CTLs induced in vitro using the above-described peptide having 5 linked epitopes of the present invention. Such pharmaceutical compositions are preferably in the form of a non-oral dosage.

5. Method of treatment

The pharmaceutical composition of the present invention can be administered to a wide range of cancer patients, for example, HLA-positive patient population selected from the group consisting of HLA-A2, HLA-A24, HLA-A26 and HLA-A3 supertypes, and treatment can be started without the need for HLA typing examination before the start of treatment.

The pharmaceutical composition of the present invention can induce and/or activate CTL epitope peptide-specific CTLs that are 2 or more, preferably 3 or more, more preferably 4 or more, and further preferably 5 CTL epitope peptide-specific CTLs contained in peptides constituting 5 linked epitopes as an active ingredient by administering the pharmaceutical composition to a cancer patient, and can induce the production of the CTL epitope peptide-specific immunoglobulin. The induction of immunoglobulin production by administration of the peptide having 5 linked epitopes contained as an active ingredient is significantly higher than that observed when CTL epitope peptides contained in the peptides having 5 linked epitopes are not linked and are each administered in admixture, and therefore, the peptide having 5 linked epitopes in the present invention can effectively treat or prevent cancer in a cancer patient.

The pharmaceutical composition of the present invention may be administered via oral administration, intravenous administration, arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, sublingual administration, intraperitoneal administration, intrarectal administration, transdermal administration, transmucosal administration, nasal administration, vaginal administration, ocular administration, inhalation administration, and the like. When preparations containing as an active ingredient a plurality of peptides having 5 linked epitopes or known CTL epitope peptides derived from tumor antigen molecules are prepared as separate pharmaceutical compositions, the pharmaceutical compositions may be administered by the same or different administration routes, simultaneously or non-simultaneously.

The administration amount of the pharmaceutical composition of the present invention may be appropriately adjusted depending on the state/severity of cancer to be treated, age, body weight and the like of each patient, but it is preferable that the pharmaceutical composition containing the peptide having 5 linked epitopes in an amount of 0.000 to 1000mg, preferably 0.00 to 100mg, more preferably 0.01 to 50mg is repeatedly administered 1 time for several days, weeks or months. In addition, in the case where CTLs induced in vitro using the peptide having 5 linked epitopes of the present invention are contained as the active ingredient of the pharmaceutical composition of the present invention, it is preferable to administer 2 × 10 at intervals of 1 week to 2 weeks per kg body weight every 1 day⁶～2×10⁸And (4) CTL.

The pharmaceutical composition of the present invention can also be administered to a cancer patient in combination with a drug generally used in cancer chemotherapy. Examples thereof include: alkylating agents such as cyclophosphamide, temozolomide and bendamustine, platinum-containing agents such as a tegafur-uracil complexing agent, a tegafur-gimeracil-oteracil potassium complexing agent, metabolic antagonists such as methotrexate and gemcitabine, platinum-containing agents such as cisplatin and oxaliplatin, plant alkaloid agents such as irinotecan, eribulin, paclitaxel, docetaxel and vincristine, anticancer antibiotics such as adriamycin, bleomycin and actinomycin D, molecular targeted therapeutic agents such as imatinib, sunitinib, gefitinib, sorafenib, everolimus, trastuzumab, bevacizumab, rituximab, cetuximab, panitumumab and モガムリズマブ, and hormonal therapeutic agents such as bicalutamide, estramustine and exemestane. These pharmaceutical products may be administered by the same or different route of administration, simultaneously or non-simultaneously, as the pharmaceutical composition of the present invention.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Examples

EXAMPLE 1 Synthesis and purification of peptide

CTL epitope peptides and peptides having 5 linked epitopes were synthesized by solid phase synthesis (Fmoc method) using a commercially available peptide synthesizer Prelude (protein technologies, Inc.). The synthetic peptides thus obtained were purified by a YMC-Pack Pro C18 column (YMC Co., Ltd.) and an HPLC system (Gilson), and stored in a cold-warm dark place after freeze-drying, and then subjected to the following examples.

The amino acid sequences of the synthesized CTL epitope peptides are shown in table 1, and the amino acid sequences of the peptides having 5 linked epitopes are shown in table 2.

WT1 (SEQ ID NO: 8) as a control peptide bound to HLA-A2 and Her2 (SEQ ID NO: 9) as a control peptide bound to HLA-A24 were synthesized.

[ Table 1]

CTL epitope peptide

Peptides	Origin of origin	Amino acid sequence	Serial number
				PEP1	Lck-246	KLVERLGAA	Sequence No. 1
PEP2	WHSC2-103	ASLDSDPWV	Sequence number 2
				PEP3	SART3-302	LLQAEAPRL	Sequence No. 3
PEP4	SART2-93	DYSARWNEI	Sequence number 4
				PEP5	SART3-109	VYDYNCHVDL	Sequence number 5
PEP6	MRP3-503	LYAWEPSFL	Sequence number 6
				PEP7	SART3-734	QIRPIFSNR	Sequence number 7
WT1	WT1-126	RMFPNAPYL	Sequence number 8
				Her2	Her2-63	TYLPTNASL	Serial number 9

[ Table 2]

Peptides having 5 linked epitopes

Peptides	Amino acid sequence	Serial number
			TPV01	PEP5-RR-PEP2-RR-PEP4-RR-PEP7-RR-PEP3	Serial number 10
TPV02	PEP5-RR-PEP2-RR-PEP7-RR-PEP3-RR-PEP4	Serial number 11
			TPV03	PEP4-RR-PEP6-RR-PEP5-RR-PEP7-RR-PEP3	Serial number 12
TPV04	PEP5-RR-PEP2-RR-PEP4-RR-PEP6-RR-PEP3	Sequence number 13
			TPV05	PEP5-RR-PEP2-RR-PEP4-RR-PEP1-RR-PEP3	Serial number 14
TPV06	PEP4-RR-PEP5-RR-PEP2-RR-PEP7-RR-PEP3	Serial number 15
			TPV07	PEP7-RR-PEP3-RR-PEP4-RR-PEP5-RR-PEP2	Serial number 16
TPV08	PEP5-RR-PEP7-RR-PEP3-RR-PEP4-RR-PEP2	Serial number 17
			TPV09	PEP6-RR-PEP1-RR-PEP4-RR-PEP5-RR-PEP2	Serial number 18
TPV10	PEP6-RR-PEP5-RR-PEP1-RR-PEP4-RR-PEP2	Serial number 19
			TPV11	PEP4-RR-PEP5-RR-PEP1-RR-PEP6-RR-PEP3	Serial number 20
TPV12	PEP7-RR-PEP2-RR-PEP4-RR-PEP5-RR-PEP3	Serial number 21
			TPV13	PEP4-RR-PEP2-RR-PEP7-RR-PEP5-RR-PEP3	Serial number 22
TPV14	PEP7-RR-PEP2-RR-PEP5-RR-PEP4-RR-PEP3	Sequence No. 23
			TPV15	PEP5-RR-PEP2-RR-PEP7-RR-PEP4-RR-PEP3	Serial number 24
TPV16	PEP5-RR-PEP2-RR-PEP4-RR-PEP3-RR-PEP7	Serial number 25
			TPV17	PEP2-RR-PEP3-RR-PEP4-RR-PEP5-RR-PEP7	Serial number 26
TPV18	PEP7-RR-PEP2-RR-PEP5-RR-PEP3-RR-PEP4	Serial number 27

In the table, "-RR-" represents arginine dimer.

Example 2 Induction of epitope peptide-specific CTL in mouse model and detection of CTL Using ELISPOT method

A peptide having 5 linked epitopes was prepared at 2mg/mL or 4mg/mL using tsukamur distilled water (tsukamur pharmaceutical factory) and filled in a B Braun Injekt syringe. After the other syringes were filled with the same amount of Incomplete Freund's Adjuvant (IFA), the two syringes were connected by a GP syringe connector, and an emulsion was prepared by thoroughly mixing a peptide solution having 5 linked epitopes and IFA. They were used in mice (HLA-A2.1 transgenic, HLA-A24 transgenic)100. mu.L each 1 time per week around the tail root of (Taconc)), for a total of 2 administrations. After 1 week from the last administration, inguinal lymph nodes were recovered from the mice. The lymph node cell suspension was prepared to 5X 10 in complete medium (RPMI-1640, 10% heat-inactivated FBS,100U/mL penicillin, 100. mu.g/mL streptomycin, 50. mu.M 2-mercaptoethanol)⁶Each cell/mL was inoculated with 1 mL/well of 24-well plates containing the target CTL epitope peptide (final concentration: 10. mu.g/mL), recombinant mouse IL-15 (final concentration: 100ng/mL), and recombinant mouse IL-21 (final concentration: 100ng/mL), respectively, at 37 ℃ with 5% CO₂Culturing in an incubator for 8 days. After 8 days, the cells were recovered and immobilized on an anti-IFN-. gamma.antibody immobilization plate included in the murine IFN-. gamma.ELISpot kit (GEN-PROBE) at 1X 10⁵Cell/well seeding. Next, spleen cells prepared from the spleens of syngeneic mice and irradiated with 30Gy of X-rays were irradiated at 1X 10⁵After cells/well were seeded in the same well as antigen presenting cells, target CTL epitope peptide or negative control peptide (final concentration: 10. mu.g/mL) was added at 37 ℃ with 5% CO₂Culturing in an incubator overnight. The next day, spots of IFN-. gamma.producing cells were visualized according to the instructions contained in the kit. The number of spots of IFN-. gamma.producing cells was quantified using an ELISPOT analyzer (Immunospot S6, Cellular Technology Ltd.). Regarding the induction of CTL specific to CTL epitope peptide, the number of IFN-. gamma.producing cells spotted in the well to which the target CTL epitope peptide was added was significantly higher than that spotted in the well to which the negative control peptide was added (Student's t-test, p)<0.05), the test piece was judged to be positive. As negative control peptides, WT1 or Her2 was used. Further, in order to visualize the CTL induction intensity, when Δ is defined as (the average number of IFN- γ -producing cell spots in the target CTL epitope peptide-added wells) - (the average number of IFN- γ -producing cell spots in the negative control peptide-added wells), Δ is set to 10 ≦ Δ<100 cases are positive, and 100. ltoreq. DELTA.<The case of 200 is indicated as a medium positive, and the case of 200. ltoreq. Δ is indicated as a strong positive.

The results of the CTL induction by CTL epitope peptide are shown in fig. 1. As shown in FIG. 1, the peptide having 5 linked epitopes of which any part has the sequence PEP5-RR-PEP2, or PEP4-RR-PEP2, or C-terminal PEP3 shows at least 3 or more kinds of CTL induction. On the other hand, the 5-linker having the same ligation order as TPV18 showed no more than 2 kinds of CTL induction.

From the above results, it is apparent that, with known CTL epitope peptides, even when a peptide having 5 linked epitopes linked via an arginine dimer is administered to a mouse, it is not necessarily possible to induce all of the epitope-specific CTLs specific to the CTL epitope peptide.

Example 3 preparation of CTL epitope peptide-immobilized beads

The peptide was immobilized on xMAP Multi-analytical COOH Microspheres (luminexcorption, hereinafter called beads) according to the following procedure. After washing the beads with MES buffer (0.1M MES-NaOH, pH7.0), the supernatant was removed by centrifugation. After repeating this twice, the beads were suspended in 75. mu.L of MES buffer. At this time, 100. mu.L of the prepared CTL epitope peptide solution (1 mg/mL) was added, and 5. mu.L of 10mg/mL EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) was further added thereto and mixed well, followed by reaction overnight at 30 ℃ in the dark. The next day, after removing the supernatant by centrifugation, 125. mu.L of 1M Tris-HCl was added and incubated at 30 ℃ for 30 minutes in the dark. After removing the supernatant, the mixture was washed twice with a washing buffer (PBS (-), 0.05% Tween20), and then suspended in an immune blocking agent (DS Pharma biomedicalal), thereby completing the immobilization of the CTL epitope peptide.

Example 4CTL epitope-specific IgG antibody titer assay

A peptide of the present invention having 5 linked epitopes was prepared at 2mg/mL with tsukamur distilled water (tsukamur pharmaceutical factory) and filled in a B Braun Injekt syringe. After the other syringes were filled with the same amount of IFA, the two syringes were connected by a GP syringe connector, and the peptide solution having 5 linked epitopes and IFA were thoroughly mixed, thereby preparing an emulsion. They were administered to CBF1 mice (C57BL/6 XBBalb/C F1) at 100. mu.L each 1 time per week around the tail root for a total of 3 times. Blood was collected from the mice 1 week after the last administration to obtain serum samples. As a control group, emulsions were prepared using respective peptide mixtures (each 1mg/mL) of PEP2, PEP3, PEP4, PEP5, PEP7, and administered on the same schedule as the peptides having the 5 epitopes linked (mixture administration group).

The CTL epitope peptide-immobilized beads diluted with the immune blocking agent were dispensed to a 96-well filter plate, washed with a washing buffer, and then a mouse serum sample diluted 200-fold with the immune blocking agent was added at 100. mu.L/well. The plate was incubated for 90 minutes in a 30 ℃ incubator with stirring at 600 rpm. After washing 3 times with the rinsing buffer, biotinylated anti-mouse IgG (H + L) (Vector laboratories) diluted 500-fold with an immuno-blocking agent was added to 100. mu.L/well, and incubated at 30 ℃ for 60 minutes with stirring at 600 rpm. After washing 3 times with the rinsing buffer, streptavidin-R-phycoerythrin conjugate (Invitrogen) diluted 500-fold with an immuno-blocking agent was added at 100. mu.L/well, and incubated at 30 ℃ for 30 minutes with stirring at 600 rpm. After washing 3 times with the rinsing buffer, the beads were suspended with the rinsing buffer added at 100. mu.L/well, and the average fluorescence intensity of the PE pigment specifically bound to each bead was measured by Bio-PlexSuspension Array System (BIO-RAD).

The results of the CTL epitope-specific IgG antibody titer measurement are shown in FIG. 2. Fig. 2 shows: an emulsion prepared by mixing equivalent amounts of IFA and tsukamur distilled water (tsukamur pharmaceutical factory) induced several-fold production of said IgG antibody in comparison with the results of measurement of the titer of a CTL epitope-specific IgG antibody in the serum of a negative control group of mice (hereinafter, simply referred to as IFA group) on the same schedule as the CTL epitope peptide mixture and the peptide having 5 linked epitopes.

As shown in fig. 2, no induction of the production of CTL epitope-specific IgG antibodies was observed in the group administered with the mixture, compared to the IFA group. On the other hand, it was confirmed that in the case of administering a peptide having 5 linked epitopes, strong induction of the production of CTL epitope-specific IgG antibodies was observed at high frequency, and furthermore, the amount of the IgG production was significantly induced by tens to hundreds of times or more by the administered peptide having 5 linked epitopes. On the other hand, in the 5-linker having the same linking sequence as in TPV17, induction of the production of a plurality of CTL epitope-specific IgG antibodies was not observed.

The above results show that: the antitumor immunity is more strongly activated by administering the peptide having 5 linked epitopes than by administering a mixture of CTL epitope peptides constituting the peptide having 5 linked epitopes.

Both induction of CTL epitope peptide-specific IgG production and induction of epitope peptide-specific CTL in cancer patients treated with the cancer peptide vaccine contribute to the effect of prolonging life. Therefore, the present invention can produce superior life-prolonging effects as compared to cancer peptide vaccine therapies composed of known CTL epitope peptides and mixtures thereof. Therefore, the peptide having 5 linked epitopes of the present invention can be preferably used as a therapeutic and/or prophylactic agent for cancer or a disease caused by cancer, or a cancer peptide vaccine, and can improve the therapeutic performance as compared with a cancer peptide vaccine therapy comprising known CTL epitope peptides and mixtures thereof.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

Claims (5)

1. A peptide having 5 linked epitopes, which is from CTL epitope peptides (a) to (g) consisting of: (a) a peptide (PEP1) represented by SEQ ID NO. 1, (b) a peptide (PEP2) represented by SEQ ID NO. 2, (c) a peptide (PEP3) represented by SEQ ID NO. 3, (d) a peptide (PEP4) represented by SEQ ID NO. 4, (e) a peptide (PEP5) represented by SEQ ID NO. 5, (f) a peptide (PEP6) represented by SEQ ID NO. 6, and (g) a peptide (PEP7) represented by SEQ ID NO. 7, wherein the 5 peptides are linked via amino acid linkers, respectively, and

the peptide having 5 linked epitopes is a sequence selected from the group consisting of sequences (a) to (p) below:

(a)PEP5-PEP2-PEP4-PEP7-PEP3；

(b)PEP5-PEP2-PEP7-PEP3-PEP4；

(c)PEP4-PEP6-PEP5-PEP7-PEP3；

(d)PEP5-PEP2-PEP4-PEP6-PEP3；

(e)PEP5-PEP2-PEP4-PEP1-PEP3；

(f)PEP4-PEP5-PEP2-PEP7-PEP3；

(g)PEP7-PEP3-PEP4-PEP5-PEP2；

(h)PEP5-PEP7-PEP3-PEP4-PEP2；

(i)PEP6-PEP1-PEP4-PEP5-PEP2；

(j)PEP6-PEP5-PEP1-PEP4-PEP2；

(k)PEP4-PEP5-PEP1-PEP6-PEP3；

(l)PEP7-PEP2-PEP4-PEP5-PEP3；

(m)PEP4-PEP2-PEP7-PEP5-PEP3；

(n)PEP7-PEP2-PEP5-PEP4-PEP3；

(o) PEP5-PEP2-PEP7-PEP4-PEP 3; or

(p)PEP5-PEP2-PEP4-PEP3-PEP7，

Wherein "-" represents an amino acid linker.

2. The peptide of claim 1 having 5 linked epitopes wherein the amino acid linker is an arginine dimer linking 2 arginines.

3. A combination of CTLs obtained by stimulating peripheral blood lymphocytes with the peptide having 5 linked epitopes according to claim 1.

4. A pharmaceutical composition for treating or preventing cancer, comprising the peptide having 5 linked epitopes according to claim 1, or the combination of CTLs according to claim 3 as an active ingredient.

5. Use of a pharmaceutical composition according to claim 4 in the preparation of an immunotherapeutic agent.