JPH08109197A - Antigen peptide for rejecting leukemic tumor - Google Patents

Abstract

PURPOSE: To obtain the leukemic tumor-rejecting antigen peptide contained in an amino acid sequence coded in an AKT gene or in an amino acid sequence coded in a non-translational region by the mutation of the AKT gene, and containing a motif bound to the molecule of MHC class I. CONSTITUTION: The new leukemic tumor-rejecting antigen peptide inducing the rejection of tumors such as cancers and containing a motif bound to the molecule of a main tissue-compatible antigen (MHC) class I comprises a peptide of formula I or II comprising an amino acid sequence coded in an AKT gene or an amino acid sequence contained as a partial sequence in an amino acid sequence coded in the non-translational region by the mutation of the AKT gene. The peptide is obtained by retrieving a motif bound to the MHC class I molecules of mouse and man among the amino acid sequences of a protein coded in the cancer gene AKT, finding out that the peptides of formulas I, II, etc., coincide with the motif, and subsequently separating the peptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a leukemia tumor rejection antigen peptide, and more particularly to a leukemia tumor rejection antigen peptide that induces rejection of tumors such as cancer.

[0002]

BACKGROUND OF THE INVENTION The immune system is a vital defense mechanism for the survival of living organisms. The first reaction of the immune response is to recognize the invading foreign body as a non-self antigen. This is because the T cell receptor, which has acquired diversity through gene rearrangement, simultaneously recognizes the antigen peptide derived from non-self presented on the cell surface and the major histocompatibility complex (MHC). It is carried out by a system that strictly discriminates self-antigens (Cell 62: 203,
1990).

Various pathways have been reported for the antigen presenting mechanism, but mainly class I (endogenous) -class II.
It is roughly divided into two pathways called the (exogenous) antigen presentation rule. The former is a processing mechanism of non-self antigens to acquire cell-mediated immunity and the latter is humoral immunity. Exogenous antigens such as bacteria are taken up by various antigen-presenting cells and then decomposed by lysosomal enzymes in endosomes, and the resulting antigenic peptides are presented to CD4 + helper T cells together with MHC class II molecules.

Helper T cells express a variety of T cell receptors acquired by gene rearrangement, and peptides derived from non-self antigens and MHC class II.
It recognizes both molecules at the same time and is activated and proliferates.
Activated helper T cells secrete various cytokines and induce B cell proliferation and differentiation (ie, production of antibodies that react with non-self antigens). The secreted specific antibody captures extracellular non-self antigens and destroys it in cooperation with the complement system.

On the other hand, endogenous antigens such as viruses, parasites and cancer antigens are expressed in almost all cells of MHC.
CD8 + killer cells (cytotoxic T with class I molecules)
Killer T cells are activated via the T cell receptor that is presented to cells: CTL) and acquired diversity as described above. Activated killer T cells are
It secretes cytotoxic factors such as lymphotoxin, tumor necrosis factor, and cell death factor Fas ligand, directly attacks target cells expressing endogenous antigens, and destroys all non-self antigens. Thus, class I (endogenous) -class I
The I (exogenous) antigen presentation rule is said to be the major non-self antigen processing mechanism.

[0006] The rapid development of the research field of immunosurveillance system in the past few years has progressed in the microsequencing method for determining the protein primary structure, and has bound to the MHC class I and class II molecules displayed on the cell surface. This is due to the fact that the structure of the antigen peptide has begun to be analyzed one after another. As a result, it was proved that a class I molecule was bound with an antigen peptide group consisting of 8 to 11 amino acid residues and a class II molecule was bound to an antigen peptide group consisting of 13 to 20 amino acid residues. Reactivity of a complex with a molecule to T cells has become the most important research subject related to autoimmune diseases, tumor immunity, rejection during transplantation, etc. (Annu. Rev. Immunol. 9:70).
7, 1991).

[0007] By the way, tumor antigens are protein molecules that appear in cells as they become cancerous, and it is considered that they are also processed in the cytoplasm and presented on MHC molecules. The existence of tumor rejection antigens was revealed about half a century ago. When a tumor induced by a chemical such as methylcholanthrene is transplanted into syngeneic mice, the tumor continues to grow and the mice die. However, even if the tumor is surgically excised after the tumor formation and the same tumor is transplanted after recovery, the tumor does not grow. That is, excision causes the host mouse to acquire resistance to its tumor. It was revealed from adoptive transfer experiments that this resistance is borne by T cells. Transplant resistance to such tumors was confirmed in many tumors, revealing the presence of rejection antigens specific to each tumor.

As described above, the chemically induced cancer has a rejection antigen unique to each tumor. The hallmark of this antigen is its great diversity. The mechanism by which diversity is generated at the molecular level is one of the top concerns of tumor immunologists, but it is still unknown. Therefore, attempts have been made to chemically extract the rejection antigen and analyze its molecule (Int. J. Cancer, 5).
4: 177-180, 1993).

For example, in a tumor rejection reaction, depending on each tumor, in other words, each tumor antigen,
It is known that CD8 or CD4 positive T cells are involved in various levels. So, in vitro
Thus, it is possible to establish a cytotoxic T cell (CTL) clone having a strong activity, and analyze the antigen specificity while maintaining this in culture for a long period of time. Such a CTL
Using a gene transfer method, several rejection antigen genes were analyzed for human melanoma to obtain a MAGE-1 gene that controls the expression of melanoma antigen MZ2-E recognized by CTL derived from autologous cancer patients ( Scien
Ce, 254: 1643, 1991). Nine peptides from the third exon of the MAGE-1 gene are targeted by CTL, and when the peptide is pulsed on the same A1-positive cells as the patient, the cells are destroyed by CTL. The frequency of Al positives corresponds to 10% of all melanoma patients. MAGE-1 is also expressed in a part of lung cancer and breast cancer, but it is not expressed in normal cells except for testis.

Furthermore, Boone et al.
-A tyrosinase was found as a human melanoma antigen presented by A2 (J. Exp. Med., 178:
489, 1993). Tyrosinase is more expected as a target for immunotherapy due to the high frequency of humans carrying HLA-A2. However, the gene is also expressed in normal melanocytes and there is concern about cross-reactivity.
For these identified genes, synthetic peptides recognized by CTL have been clarified, but it has not yet been reported whether these peptides can actually perform active immunization. The tumor rejection antigens reported to date relate to highly immunogenic melanomas, and there are no reports of leukemia tumor rejection antigens.

[0011]

An object of the present invention is to provide a leukemia tumor rejection antigen peptide. Tumor rejection is caused by the local interaction of 8-11 residue peptides on MHC molecules. However, most of these peptides are extremely small in the living body, and due to the lack of an effective analytical method, most of them are unknown regarding tumor immunity in leukemia cells. Development of an antigen has been desired. Based on such a situation, the present inventors have used mouse leukemia cells as a model in order to efficiently find leukemia tumor rejection antigen peptides, and examined the tumor rejection antigen mechanism in detail, as well as leukemia diseases. As a result of intensive research aimed at establishing treatments and diagnostic methods for various pathological conditions, the present invention succeeded in identifying a peptide involved in a novel leukemia tumor rejection antigen, and completed the present invention. .

[0012]

According to the present invention, an AKT
Amino acid sequence encoded by a gene or the AKT
A peptide composed of an amino acid sequence contained as a partial sequence in an amino acid sequence encoded by its untranslated region due to mutation of a gene, and having a motif that binds to a major histocompatibility complex (MHC) class I molecule. There is provided a leukemia tumor rejection antigen peptide comprising.

Preferred embodiments of the present invention are as follows. (1) The leukemia tumor rejection antigen peptide, wherein the peptide is composed of 15 or less amino acid residues. (2) The leukemia tumor rejection antigen peptide, wherein the peptide is composed of 8 to 11 amino acid residues. (3) The leukemia tumor rejection antigen peptide, wherein the AKT gene is mouse v-AKT, mouse c-AKT, human v-AKT, or human c-AKT gene. (4) The leukemia tumor rejection antigen peptide as described above, wherein the amino acid sequence encoded by the mouse v-AKT gene is the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. (5) The leukemia tumor rejection antigen peptide, wherein the amino acid sequence encoded by the mouse c-AKT gene is the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. (6) The above leukemia tumor rejection antigen peptide, wherein the amino acid sequence encoded by the untranslated region due to the mutation of the AKT gene is an amino acid sequence obtained when the untranslated region is translated into amino acids due to the mutation of the c-AKT gene. . (7) The above leukemia tumor rejection antigen peptide, wherein the amino acid sequence obtained when the untranslated region of the mouse c-AKT gene is mutated and translated into amino acids is the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. (8) The peptide is Ile Pro Gly Le
u Pro Leu Ser Leu (IPGLPL
SL) a leukemia tumor rejection antigen peptide. (9) The above leukemia tumor rejection antigen peptide, wherein IPGLPLSL exhibits activity in leukemia cells having mouse H-2Ld as an MHC class I molecule. (10) The peptide is Met Tyr Glu M
et Met CysGly Arg Leu (MY
The leukemia tumor rejection antigen peptide which is EMMCGRL). (11) The leukemia tumor rejection antigen peptide as described above, wherein MYEMMCGRL is active in leukemia cells having mouse H-2Kd as an MHC class I molecule.

The present invention will be described in detail below. The tumor rejection antigen peptide of the present invention is a polypeptide in which methionine is not bound to the N-terminus of the amino acid sequence of the peptide,
And an intermediate in which part or all of the signal peptide is bound or deleted at the N-terminal of the above amino acid sequence. It is possible to change a part of the structure of the DNA encoding the polypeptide by natural mutation or artificial mutation without changing the main activity of the polypeptide. The tumor rejection antigen polypeptide of the present invention also includes a polypeptide having a structure corresponding to a homologous variant having the above amino acid sequence. The tumor-rejection antigen peptide of the present invention is a protein molecule that appears in cells with canceration, undergoes processing in the cytoplasm, and becomes MHC.
It is presented on the molecule.

Since the tumor rejection antigen peptide of the present invention is bound to the MHC class I molecule by non-covalent bond,
The acid treatment easily separates from the MHC molecule. This short peptide having a molecular weight of several thousand daltons or less can be separated from a protein molecule having a molecular weight of 5 KD or more by a molar cut. The peptide-containing extract can be subjected to high performance liquid chromatography (HPLC) using a reverse phase column to examine the elution pattern associated with the acetonitrile gradient. Each peak of these eluted peptides can be collected and pulsed to another tumor corresponding to MHC, which is used as a target cell, and the sensitizing activity of killer T cells can be examined by the ⁵¹ Cr release method.
The peptide whose activity has been detected can be determined for its amino acid sequence using the Edman degradation method and further using a tandem mass spectrometer. In this way, cancer antigen peptides recognized by T cells can be identified.

However, in practice, determination of the amino acid sequence by the Edman degradation method or the tandem mass spectrometer requires a peptide amount of at least several tens of picomoles, which results in an enormous number of tumor cells. Will be needed. Another problem is that there are at least several types of peptides contained in each fraction eluted by HPLC, and therefore, there are several types of signals obtained by the Edman degradation method, and it is difficult to accurately determine the amino acid sequence. .
For this reason, a method of reading the amino acid sequence from how the peptide breaks using a tandem mass spectrometer is drawing attention, but this method is not easy to accurately predict the amino acid sequence from the data actually obtained. Therefore, the present inventors have found that BALB / c radiation leukemia RL♂1 (hereinafter, RL
(Abbreviated as 1) based on the existence of a unique antigen recognized by CTL specific to cells (Proc. Natl.
Acad. Sci. USA, 76: 3486, 197.
9) As a result of earnest studies on tumor immunity, they succeeded in obtaining a leukemia tumor rejection antigen.

The following is a description of how the present inventors discovered the leukemia tumor rejection antigen. Inoculation of radiation-induced mouse leukemia RL1 into F1 of BALB / c and B6 mice results in rejection after once forming tumors. Administration of anti-CD8 antibody to host mice in vivo ( in vivo )
Depletion of D8 T cells blocks rejection and kills all host mice. On the other hand, the anti-CD4 antibody is not affected. For this reason, rejection is mainly due to CD
It can be seen that 8-positive T cells are involved. Restimulation of tumor-rejected mouse splenocytes with RL1 cells in vitro induces tumor-specific cytotoxic T cells (CTL) (Proc. Natl. Acal. Sci. US).
A 76: 3486-3490, 1979). Inoculation of BALB / c nu / nu mice with induced CTL and then inoculation of these mice with RL1 blocks proliferation of RL1 cells (Immunology 56: 1.
41-151, 1985).

For the purpose of clarifying the rejection antigen peptide recognized by such CTLs responsible for the rejection of RL1 cells, the present inventors attempted to establish CTLs from splenocytes of tumor-rejected mice. , 6 types of cloned CTL strains were established. As a result of cytotoxic activity measurement, it was found that bulk CTL and all 6 CTL clones specifically injured RL1 cells (Table 1).

[0019]

[Table 1] (Footnote) The specificity of the cytotoxic activity of RL1 CTL cells obtained in Example 1 to target cells (RL1 and P815) is shown. As CTL cells, bulk CTL for RL1 and 6 types of cloned CTL cells (clone) were used.
12, 14, 31, 33, 44, 103) was used. Ratio of cytotoxic T cells (CTL): target cells = 9: 1

From the results shown in Table 1, it can be seen that specific antigens recognized by these CTLs are present in RL1 cells. Further, as shown in Table 2, as a result of the cytotoxicity inhibition test using the anti-H-2 monoclonal antibody, it was found that the cytotoxicity of these CTLs was all Ld-restricted. Suppression of cytotoxic activity occurs with anti-CD8 antibody, and CTL are CD8-positive T cells. That is, it was found that the T cell receptors of these CTLs recognize the peptide bound to the Ld molecule.

[0021]

[Table 2] (Footnote) It is a cytotoxicity inhibition test by a monoclonal antibody against the mouse major histocompatibility antigen (H-2) obtained in Example 1. Mouse major histocompatibility antigens include K, D and L as class I molecules and A and E as class II molecules. Serum and monoclonal antibody were used at 50-fold dilution. Bulk to cloned cytotoxic T cell (CTL): RL♂1 target cell ratio = 5: 1. *: Normal mouse serum

The present inventors, as shown in FIG.
Treatment of cells with acid (trifluoroacetic acid: TFA) to MH
The C-linked peptide was extracted. Fractions with a molecular weight of 5000 or less were fractionated by reverse phase HPLC (ODP column), and each fraction was added to mouse cytoma p815 cells derived from DBA / 2 expressing the same H-2 molecule (H-2d) as RL1. , Sensitizing activity was investigated. If p815 cells become cytotoxic by RL1-specific CTL in the same manner as RL1 cells, it means that the fraction used for sensitization contains the target antigen peptide. 12
Molecular weight of RL1 ascites cells obtained from a single cancer-bearing mouse
20-60% of TFA extract of 0 or less in the presence of TFA
Acetonitrile gradient elution was performed.

As shown in FIG. 1, strong sensitizing activity was observed in the fractions at 23 minutes (peak a) and 26 minutes (peak b), but not in the other fractions. The sensitizing activity of these fractions was
All were similarly observed for L and 6 CTL clones. From this result, it was found that the extracted antigen was the major CTL recognition antigen. As a result of investigating whether or not p815 sensitized with these active fractions suppresses cytotoxic activity against RL1 labeled with ⁵¹ Cr, sensitized p81 was obtained.
5 suppressed the cytotoxic activity like the unlabeled RL1.

From these results, it is understood that the RL1CTL recognition antigen is relatively single and is efficiently extracted with acid. The sensitizing activity of the fraction, like the cytotoxic activity on RL1, is inhibited by anti-Ld antibody,
It is also inhibited by known Ld-binding peptides (Table 2).
These indicate that the activity of the fraction is due to the peptide that binds to the Ld molecule. RL
When one cell is solubilized, the Ld molecule is treated with TFA with an affinity column, and the liberated substance having a molecular weight of 5000 or less is fractionated by the reverse phase HPLC using the ODP column described above,
The active fraction was obtained at 23 minutes, which was the same as in the case of acid extraction directly from RL1 cells. In this case, almost no sensitizing activity was observed in the 26-minute fraction.

Therefore, in order to purify and identify the active peptide, 2.5 × 10 ^{11 was obtained} from 250 RL1 tumor-bearing mice.
Ascites cells were collected, acid-extracted with TFA, and
Reversed-phase HPLC was repeatedly carried out using a DP column, and 23-minute and 26-minute sensitizing fractions were pooled. For each pooled active fraction, C2 / C
Rechromatography was performed on 18 columns under neutral and acidic conditions to obtain a unimodal peak. The primary structure was analyzed by the Edman decomposition method, and the
From the activity peak derived from minute, octamer pRLla
(Amino acid sequence; Ile Pro Gly Leu P
ro Leu Ser Leu, or IPGLPLS
L) is from the activity peak derived from 26 minutes, p
RL1b (amino acid sequence; Ser Ile Ile P
ro Gly Leu Pro Leu Ser Le
The sequence of the u, ie SIIPGLPLSL) peptide was obtained.

Synthetic peptides based on these sequences were eluted by ODP reverse-phase HPLC at the same 23 and 26 minutes as the peptide processed in the cell, at 1-100 n.
It was confirmed that M concentration showed sensitizing activity. From the sequence information of the homology search, it was revealed that these two types of peptides are part of the protein encoded by the oncogene AKT-1 gene. From this, pRL1
It is considered that a is formed by cutting two amino acids of pRL1b to form one leukemia tumor rejection antigen epitope.

The origin of the oncogene AKT having homology with the leukemia tumor rejection antigen peptide obtained in the present invention is as follows. AKT8 virus is a retrovirus taken from T cell lymphoma (Pro.
c. Natl. Acad. Sci. U. S. A. 74,
3065, 1977), which has the activity of transforming cultured mink lung cells. The v-AKT gene is AKT8
A gene cloned from a virus-infected mink lung cell, which is a retrovirus-derived Gag.
Protein and untranslated region of cell-derived c-AKT (c-AK
The translated protein of the ATG codon upstream of T (60 residues) and c
-It has been reported that the AKT protein is a fused protein (Science, 254, 274-277).

Regarding c-AKT, it has been cloned in mouse and human, and there is 90% homology at the nucleic acid level and 98% homology at the amino acid level between human and mouse (Proc. Natl. Ac.
ad. Sci. USA, 89, 9267-9271, 1
992). Regarding the c-AKT gene, s
There is a rc homology 2-like domain and a protein kinase-like domain on the C-terminal side.

The peptides identified according to the invention are c
-Corresponding to the untranslated region upstream of the ATG codon of the AKT gene, which is derived from v-AKT or the c-AKT gene is a natural mutation or a mutation caused by canceration (point mutation, deletion, insertion) , Frame shift, etc.). Table 3 shows the comparison results of the amino acid sequences of the leukemia tumor rejection antigen peptide and the AKT gene peptide.

[0030]

[Table 3]

The leukemia tumor rejection antigen peptide of the present invention is
In the mouse allotype H-2Ld of major histocompatibility complex (MHC) class I molecule, as shown in Table 3, IPG
LPLSL. However, other major histocompatibility antigens are not limited to this amino acid sequence. MHC class I molecules are present on H-2K, D, and L on chromosome 17 in mice, and depending on the type of mouse, b, d, f, j, k, p, q, r, s, and u. , V,
Halotypes such as z are known. On the other hand, in humans, M
HC class I molecules are HLA-A, B, and
HLA-A is present on C and due to individual differences in humans,
25 types or more, HLA-B, 32 types or more, HLA
In -C, 11 or more types have been reported.

Several reports have recently been made on the analysis of peptides that bind to these MHC class I molecules (Annu. Rev. Immunol. 12: 1.
81-207, 1994). It immunoprecipitates mouse and human class I molecules from solubilized cell membranes,
This was done by purifying self-peptides self-binding to these class I molecules, performing amino acid analysis and analyzing their primary structure. As a result, it has been clarified that the motif of the peptide that binds to each class I molecule is different for each class I molecule encoded by a different locus, that is, for each halotype.

For example, in mouse and H-2Ld, P is present at the position of the second amino acid from the N-terminus and C-terminal (8-1).
A peptide having F or L is bound to the first position). The H-2Ld-binding leukemia tumor rejection antigen peptide of the present invention, IPGLPLSL, also matches this motif. In addition, mouse, H-2Kd (2nd; Y, C terminal; I,
L, V), H-2Kk (second; E, C terminal; I), H
-2Dd (2nd; G, 3rd; P, C terminal: L, I,
F), H-2Db (5th; N, C termini; M, I), etc., and also for humans HLA-A2. l (2nd; L, M, I, C-terminal; V, L, I, A), HLA-
A3.1 (2nd: L, C terminal; Y, K), HLA-B
7 (2nd; P, C terminal; L, I) and the like have been reported (Annu. Rev. Immunol. 12; 181-).
207, 1994).

Therefore, the leukemia tumor rejection antigen peptide of the present invention includes the above-mentioned mouse and human MHC class I amino acid sequences of the protein encoded by the oncogene AKT.
The motif is not particularly limited as long as it is a motif that binds to a molecule, and can be used as a tumor rejection antigen peptide. The present inventors have actually confirmed that the mouse H-2Kd
The second Y, C-terminal I, L, and V sequences, which are motifs of, were searched for with v-AKT (SEQ ID NO: 1), and GY
KERPQDV (58th-66th), DYLHSEKN
V (283-291st), MYEMMCGRL (36
0-368th), FYNQDHEKL (370-37)
The 8th) peptide was found to match this motif.

Then, using the bulk CTLs sensitized with these peptides, the cytotoxic activity against p815 cells having H-2Kd was examined, and it was found that MYEMMCGRL
It was found that (360-368th) is an active peptide. This peptide also corresponds to the 339-347th peptide of mouse c-AKT (SEQ ID NO: 2). Therefore, MHC class I molecules from other mice,
For human MHC class I molecules, leukemia tumor rejection antigen peptides can be efficiently identified in the same manner.

[0036]

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

Example 1 (1)Cytotoxic activity measurement The cytotoxic activity in the present invention is measured as follows.
I went. Tumor cells (target cells) 2 × 10⁶2M
B_qNa ₂ ⁵¹CrO_FourAnd 0.3 ml of culture medium for 1 hour
Cultured and labeled. Wash these cells and
I used it. 1x10 labeled^FourIndividual cells (100
CTL cells (100 μl) were used as target cells.
l) was added as an effector cell, and 96-well culture pre
It was cultured in an autoclave. Blocking assay using antibody
Is 100 μl of diluted monoclonal antibody
Target cells labeled with 50 μl of target cells
Added and went.

The stimulation test using the HPLC fraction was carried out by diluting 5 to 10 μl of the HPLC fraction into 100 μl of the culture medium to obtain 1 × 10 ⁴ labeled target cells (5
0 μl) for 1 hour at room temperature and 50 μl of effector cells were added. After incubation for 4 hours, the culture supernatant (100 μl) was taken and the radioactivity was measured with a gamma counter. The specific damaging activity was calculated as follows. CTL activity = [(ab) / (cb)] × 100 a: radioactivity released when target cells are incubated with effector cells b: spontaneous release radioactivity released when incubated with effector cells only c: Maximum free radioactivity generated when target cells are treated with a surfactant or the like

(2) RL1 cell-specific cytotoxic T cells
RL1 1 × 10 ⁶ cells which were established with maintaining CB6F1 mouse splenocytes 5 × 10 ⁷ cells and irradiated (100 Gy) of were cultured for 5 days in flasks for cell culture. The culture medium is RPMI 1
640, 10% fetal calf serum (FCS), 2 mM glutamic acid, 100 u / ml penicillin, 100 μg / ml streptomycin, 50 mM 2-mercaptoethanol were used. Cells detached from the culture flask (5 x 10 ⁴
Cells) as bulk CTL cells, 1 × 1 per week
0 as ^five irradiated with (100 Gy) were RL1 cells stimulated cells, 5 × 10 ⁵ cells of irradiated (20 Gy) spleen cells feeder cells CB6F1 that, 5n
It was maintained in a culture medium supplemented with g / ml human recombinant II-2. To clone CTL cells, seed 96-well plates with 3 to 0.3 CTL cells and 1 ×.
10 ⁴ irradiated RL1 cells and 1 × 10 ⁶ irradiated CB6F1 splenocytes were cultured in the presence of IL-2. By examining the cytotoxic activity of the cells that had proliferated after 10 to 14 days, 6 types of cloned CTL cells could be obtained by cloning.

Table 1 shows the cytotoxic activity of the obtained bulk CTL cells, cloned CTL cells against RL1 cells and p815 cells. Mouse major histocompatibility antigen (H-
Table 2 shows the results of the cytotoxicity inhibition test with various monoclonal antibodies against 2). Mouse major tissue antigens include K, D, and L as class I molecules and A and E as class II molecules, but among these monoclonal antibodies, only anti-Ld molecule monoclonal antibody Since the leukemic tumor rejection antigen is suppressed, the leukemic tumor rejection antigen is found to be bound to the Ld molecule.

Example 2 The leukemia tumor rejection antigen peptide was purified by the following method. (1) Balb / c mouse 25 containing 2.5 × 10 ¹¹ purified RL1 cells by high performance liquid chromatography
Prepared from 0 ascites fluids. The cells obtained are 0.1% T
It was homogenized for 10 strokes with a dance homogenizer in FA (trifluoroacetic acid) and treated with a sonicator for 3 minutes. The homogenate was 0.1% TFA (pH 2.
Stirred at 0) for 30 minutes. After centrifugation at 10,000 G for 30 minutes, the supernatant was filtered with a molecular cut membrane (molecular weight 5000, Milliboa). The filtrate obtained from 10 mice was dissolved in 0.1% TFA and subjected to reverse phase high performance liquid chromatography (HPLC) (ODP; 10 × 25).
0 mm, 10 μm, ASAHIPAK, KAWASAK
Separated in I). The column elution solvent was a gradient of acetonitrile from 20% to 60% in 0.1% TFA.
The fraction was fractionated in 2 ml portions. The activity of each fraction was measured using killer T cells against RL1 cells,
As target cells, p815 cells having H-2Ld were used. FIG. 1 shows the results of the first C18 column. The fractions of peak a and peak b had cytotoxic activity against p815 cells.

In addition, for purification, reverse phase HPLC
C2 / C18 column (SuperPac ^™ Pep-
S; 4 × 250 mm, 5 μm particles; P
The fractions of peak a and peak b were applied to Harmacia LKB, Swerden) and eluted under a neutral condition with an acetonitrile concentration gradient at a rate of 1 ml / min. Reversed-phase HPLC fractionated profile and aliquots of each fraction were added to p815 cells and
The results of examining the damaging activity by 1-specific CTL are shown in FIG. FIG. 2A is a profile when a fraction derived from peak a was applied, and strong cytotoxic activity was observed at 12 minutes (peak a ′), and FIG. 2B is a profile when a fraction derived from peak b was applied. 29 minutes in profile (peak b ')
A strong cytotoxic activity was observed in the fraction.

FIG. 2 The fractions of peak a ′ and peak b ′ were analyzed by reverse phase HPLC (C2 / C18 column, 0.1% TFA under acidic condition, acetonitrile concentration gradient; 0.5%) for the third time.
(A) or 1.66% (B) / min, 2 ml / mi
The profile fractionated in m) is shown in FIG. A part of each fraction was taken and added to p815 cells, and the RL1-specific CTL-damaging activity was examined. FIG. 3A shows peak a ′.
The profile when the fractions derived from
Strong cytotoxic activity was observed at 5 minutes (peak a ″), and FIG.
B is the profile when the fraction derived from peak b ′ was applied, and a strong cytotoxic activity was observed in the fraction at 23 minutes (peak b ″).

(2) Peptide Sequence The peptide sequence was determined by the Edman degradation method using a peptide sequencer (Model 477A; Applied Biosystems). The peak a ″ fraction is IPGLPLSL and the peak b ″ fraction is SIIPG
Determined to be LPLSL.

Example 3 Characterization of Binding Peptide (1) Peptide Synthesis The peptides of IPGLPLSL and SIIPGLPLSL were synthesized by a solid phase synthesis method using the F-moc method, using a peptide synthesizer (Model 430A, Applied Biosystems). ) Was used for chemical synthesis. The synthesized peptide was a reversed-phase HPLC column (C8 column, 10 × 100 mm, 20
μm Particle size, Applied Biosystems), and purified to 98% or more by acetonitrile concentration gradient in the presence of 0.1% TFA acetonitrile. The results of examining the CTL stimulating activity using these peptides are shown in FIG.

Using the cytotoxic activity of RL1 CTL cells as target cells, T1.1.1 (Ld-transfected L cells); T4.8.3 (Db-transfected L cells); p815 (Ld-expression) Cell), the effect of peptide addition was examined. As a peptide,
pRLIA (IPGLPLSL) and pRL1b (SII
PGLPLSL) was examined by ⁵¹ Cr release capability by addition, PRL1b and pRL1a (IPGLPLSL)
(SIIPGLPLSL) T1.1.1 and p815 cells, both of which express H-2Ld, showed cytotoxic activity, but T expressing H-2Db.
This activity was not observed in 4.8.3 cells. Thus, the peptide is a tumor rejection antigen that specifically binds to H-2Ld molecules. pRL1b also pRL
It had the same activity as that of 1a because pRL1b was decomposed by serum or the like in the culture medium and changed to pRL1a.

[Example 4] (1) When the second Y- and C-terminal L, L, and V sequences of the mouse and H-2Kd motifs were searched by v-AKT, GYKERPQDV (58-66th position) ), DY
LHSEKNV (283-291st), MYEMMC
GRL (360-368th), FYNQDHEKL
It was found to be the peptide (370-378th position). This peptide was chemically synthesized by a solid phase synthesis method using the F-moc method using a peptide synthesizer (Model 430A, Applied Biosystems). The synthesized peptide was a reverse phase HPLC column (C8 column, 10 × 1).
00 mm, 20 μm Particle size, Applied Biosystems), 0.1% TFA
Purification was performed to 98% or more by a gradient of acetonitrile concentration in the presence of acetonitrile.

(2) 3 × 1 splenocytes of BALB / c mouse
0 ⁶ and the synthesized peptide were adjusted to 1 × 10 ⁻⁴ M each and cultured on a cell culture plate for 5 days. The culture medium is RPMI 1640, 10% fetal calf serum (FC
S) 2 mM glutamic acid, 100 μ / ml penicillin, 100 μg / ml streptomycin, 50 mM2
-Mercaptoethanol was used. Cells (5 × 10 ⁵ ) were collected from the culture plate and used as bulk CTL cells in the cytotoxic activity test. When the cytotoxic activity against p815 cells (target cells) having H-2Kd sensitized with these peptides was examined (FIG. 5), MEYEMM
It was found that CGRL (360-368th position) was an active peptide when 1 × 10 ⁻⁴ M was added.

[0049]

INDUSTRIAL APPLICABILITY By using the leukemia tumor rejection antigen peptide of the present invention, a technique for elucidating diagnostic and therapeutic methods for various pathological conditions is provided. In particular, it is effective for tumor immunotherapy of leukemia. INDUSTRIAL APPLICABILITY The tumor rejection antigen peptide of the present invention is effective in elucidating the mechanism of tumor rejection of the immune system, diagnosing and treating tumor immune diseases, and developing immune activators. In addition to this, the oncogene AKT is associated with gastric cancer cells (Pr
oc. Natl. Acad. Sci. USA, vol.
84, 5034-5037, 1987) and ovarian cancer (Pr.
oc. Natl. Acad. Sci. USA, vol.
89, 1992), it is also useful for elucidation of the mechanism of carcinogenesis and diagnosis and treatment of cancer.

The treatment of the leukemia tumor rejection antigen peptide of the present invention in humans will be described below with reference to examples. First, the leukemia tumor rejection antigen peptide encoded by the AKT gene that binds to the MHC class I molecule of each patient is selected. Next, the leukemia tumor rejection antigen peptide is added to the patient's tumor cells or cells having the same MHC class I molecule. The patient's T cells are then added and cultured in vitro to specifically induce cytotoxic T cells that recognize the leukemia tumor rejection antigen peptide and returned to the patient for treatment. In addition to this, the leukemia tumor rejection antigen peptide can be administered alone or in combination with various drugs that increase stability, such as liposomes. (In this specification, when an amino acid or the like is represented by an abbreviation, an abbreviation according to IUPAC-IUB or an abbreviation commonly used in the art is used.)

[0051]

[Sequence list]

SEQ ID NO: 1 Sequence length: 501 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Arg Glu Glu Thr Leu Ile Ile Ile Pro Gly Leu Pro Leu Ser Leu 1 5 10 15 Gly Ala Thr Asp Thr Met Asn Asp Val Ala Ile Val Lys Glu Gly Trp 20 25 30 Leu His Lys Arg Gly Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe 35 40 45 Leu Leu Lys Asn Asp Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln 50 55 60 Asp Val Asp Gln Arg Glu Ser Pro Leu Asn Asn Phe Ser Val Ala Gln 65 70 75 80 Cys Gln Leu Met Lys Thr Glu Arg Pro Arg Pro Asn Thr Phe Ile Ile 85 90 95 Arg Cys Leu Gln Trp Thr Thr Val Ile Glu Arg Thr Phe His Val Glu 100 105 110 Thr Pro Glu Glu Arg Glu Glu Trp Ala Thr Ala Ile Gln Thr Val Valala 115 120 125 Asp Gly Leu Lys Arg Gln Glu Glu Glu Thr Met Asp Phe Arg Ser Gly 130 135 140 Ser Pro Ser Asp Asn Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala 145 150 155 160 Lys Pro Lys His Arg Val Thr Met Asn Glu Phe Glu Tyr Leu Lys Leu 165 170 175 Leu Gly Lys Gly Thr Phe Gly Lys Val Ile Leu Val Lys Glu Lys Ala 180 185 190 Thr Gly Arg Tyr Tyr Ala Met Lys Ile Leu Lys Lys Glu Val Ile Val 195 200 205 Ala Lys Asp Glu Val Ala His Thr Leu Thr Glu Asn Arg Val Leu Gln 210 215 220 Asn Ser Arg His Pro Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr 225 230 235 240 His Asp Arg Leu Cys Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu 245 250 255 Phe Phe His Leu Ser Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg 260 265 270 Phe Tyr Gly Ala Glu Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu 275 280 285 Lys Asn Val Val Tyr Arg Asp Leu Lys Leu Glu Asn Leu Met Leu Asp 290 295 300 Lys Asp Gly His Ile Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu Gly 305 310 315 320 Ile Lys Asp Gly Ala Thr Met Lys Thr Phe Cys Gly Thr Pro Glu Tyr 325 330 335 Leu Ala Pro Glu Val Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp 340 345 350 Trp Trp Gly Leu Gly Val Val Met Tyr Glu Met Met Cys Gly Arg Leu 355 360 365 Pro Phe Tyr Asn Gln Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met 370 375 380 Glu Glu Ile Arg Ph e Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu 385 390 395 400 Leu Ser Gly Leu Leu Lys Lys Asp Pro Thr Gln Arg Leu Gly Gly Gly 395 410 415 Ser Glu Asp Ala Lys Glu Ile Met Gln His Arg Phe Phe Ala Asn Ile 420 425 430 Val Trp Gln Asp Val Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys Pro 435 440 445 Gln Val Thr Ser Glu Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe Thr 450 455 460 Ala Gln Met Ile Thr Ile Thr Pro Pro Asp Gln Asp Asp Ser Met Glu 465 470 475 480 Cys Val Asp Ser Glu Arg Arg Pro His Phe Pro Gln Phe Ser Tyr Ser 485 490 495 Ala Ser Gly Thr Ala 500

SEQ ID NO: 2 Sequence length: 479 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Asn Asp Val Ala Ile Val Lys Glu Gly Trp Leu His Lys Arg Gly 1 5 10 15 Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe Leu Leu Lys Asn Asp 20 25 30 Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln Asp Val Asp Gln Arg 35 40 45 Glu Ser Pro Leu Asn Asn Phe Ser Val Ala Gln Cys Gln Leu Met Lys 50 55 60 Thr Glu Arg Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp 65 70 75 80 Thr Thr Val Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu Glu Arg 85 90 95 Glu Glu Trp Ala Thr Ala Ile Gln Thr Val Ala Asp Gly Leu Lys Arg 100 105 110 Gln Glu Glu Glu Thr Met Asp Phe Arg Ser Gly Ser Pro Ser Asp Asn 115 120 125 Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala Lys Pro Lys His Arg 130 135 140 Val Thr Met Asn Glu Phe Glu Tyr Leu Lys Leu Leu Gly Lys Gly Thr 145 150 155 160 Phe Gly Lys Val Ile Leu Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr 165 170 175 Ala Met Lys Ile Leu Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val 180 185 190 Ala His Thr Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro 195 200 205 Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys 210 215 220 Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu Ser 225 230 235 240 Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg Phe Tyr Gly Ala Glu 245 250 255 Ile Val Ser Ala Leu Asp Tyr Leu His Ser Glu Lys Asn Val Val Tyr 260 265 270 Arg Asp Leu Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile 275 280 285 Lys Ile Thr Asp Phe Gly Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala 290 295 300 Thr Met Lys Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val 305 310 315 320 Leu Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly 325 330 335 Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln 340 345 350 Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg Phe 355 360 365 Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu Ser Gly Leu Leu 370 375 380 Lys Lys Asp Pro Th r Gln Arg Leu Gly Gly Gly Ser Glu Asp Ala Lys 385 390 395 400 Glu Ile Met Gln His Arg Phe Phe Ala Asn Ile Val Trp Gln Asp Val 395 410 415 Tyr Glu Lys Lys Leu Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu 420 425 430 Thr Asp Thr Arg Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr 435 440 445 Ile Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu Arg 450 455 460 Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Gly Thr Ala 465 470 475

SEQ ID NO: 3 Sequence length: 94 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Asp Gln Arg Thr Asp Arg Ala Ala Ser Cys Gly Arg His Arg Gly 1 5 10 15 Gly Pro Asp Pro Ala Ser Ser Ala Arg Pro Asp Ala Ala Ala Phe Ser 20 25 30 Arg Pro Arg Pro Ala Pro Ala Arg Gly Met Arg Ser Gly Gly Arg Pro 35 40 45 Arg Pro Arg Pro Gly Xaa Ala Gln Ser Pro Ala Arg Gly Gly Pro Thr 50 55 60 Leu Arg Pro Gly Arg Leu Gly Ser Ala Tyr Arg Glu Glu Thr Leu Ile 65 70 75 80 Ile Ile Pro Gly Leu Pro Leu Ser Leu Gly Ala Thr Asp Thr 85 90

[Brief description of drawings]

FIG. 1 is a diagram showing a result of a reverse phase HPLC separation pattern of a first RL1 antigen peptide and a cytotoxic activity test.
Strong cytotoxic activity was observed in the fractions at 23 minutes (peak a) and 26 minutes (peak b).

FIG. 2 is a diagram showing a result of a reverse phase HPLC separation pattern of a second RL1 antigen peptide and a cytotoxic activity test.
FIG. 2A is a profile when the fraction derived from peak a was applied, and a strong cytotoxic activity was observed in the fraction at 12 minutes (peak a ″). In FIG. 2B, the fraction derived from peak b was applied. In the profile at the time, strong cytotoxic activity was observed in the fraction at 29 minutes (peak b ″).

FIG. 3 is a diagram showing a result of a reverse phase HPLC separation pattern of a third RL1 antigen peptide and a cytotoxic activity test.
FIG. 3A is a profile when the fraction derived from peak a ′ was applied, and a strong cytotoxic activity was observed in the fraction at 24.5 minutes (peak a ″). FIG. 3B shows that the fraction derived from peak b ′. The profile when applying minutes, 2
Strong cytotoxic activity was observed in the fraction at 3 minutes (peak b ″).

FIG. 4: Peptide fragment pR obtained in Example 3
L1a (IPGLPLSL) and pRL1b (SIIPG
It is a figure which shows the CTL stimulating activity of LPLSL).

FIG. 5 is a diagram showing the results of a cytotoxic activity test of an antigenic peptide against Kd. The vertical axis represents the cytotoxic activity (%),
The horizontal axis represents the ratio of effector cells to target cells in E / T ratio. It can be seen that when KD3 (MYEMMCGRL) is added to p815 cells, the cytotoxic activity is increased.

Claims (12)

[Claims] 1. A peptide composed of an amino acid sequence encoded by the AKT gene or an amino acid sequence contained as a partial sequence in the amino acid sequence encoded by the untranslated region of the AKT gene by mutation, A leukemia tumor rejection antigen peptide comprising a motif that binds to a major histocompatibility complex (MHC) class I molecule. 2. The leukemia tumor rejection antigen peptide according to claim 1, wherein the peptide is composed of 15 or less amino acid residues. 3. The leukemia tumor rejection antigen peptide according to claim 1, wherein the peptide is composed of 8 to 11 amino acid residues. 4. The AKT gene has mouse v-AKT, mouse c-AKT, human v-AKT, or human c-AK.
The leukemia tumor rejection antigen peptide according to any one of claims 1 to 3, which is a T gene. 5. The leukemia tumor rejection antigen peptide according to claim 4, wherein the amino acid sequence encoded by the mouse v-AKT gene is the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. 6. The leukemia tumor rejection antigen peptide according to claim 4, wherein the amino acid sequence encoded by the mouse c-AKT gene is the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing. 7. The amino acid sequence encoded by the untranslated region due to mutation of the AKT gene is an amino acid sequence obtained when the untranslated region is translated into amino acids due to mutation of the c-AKT gene. Leukemia tumor rejection antigen peptide. 8. The leukemia according to claim 7, wherein the amino acid sequence obtained when the untranslated region of the mouse c-AKT gene is mutated and translated into an amino acid is the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing. Tumor rejection antigen peptide. 9. The peptide is Ile Pro Gl
y Leu ProLeu Ser Leu (IPG
LPLSL), The leukemia tumor rejection antigen peptide according to any one of claims 1 to 8. 10. IPGLPLSL is MHC class I
The leukemia tumor rejection antigen peptide according to claim 9, which exhibits activity in leukemia cells having mouse H-2Ld as a molecule. 11. The peptide is Met Tyr G.
lu Met Met Cys Gly Arg Le
The leukemia tumor rejection antigen peptide according to any one of claims 1 to 8, which is u (MYEMMCGRL). 12. The leukemia tumor rejection antigen peptide according to claim 11, wherein MYEMMCGRL exhibits activity in leukemia cells having mouse H-2Kd as an MHC class I molecule.