# Patent

## Title
HLA-binding peptide, precursor thereof, DNA fragment and recombinant vector encoding the same

## Abstract

There is provided an HLA-binding peptide being excellent in binding affinity to an HLA-A type molecule. An HLA-binding peptide capable of binding to an HLA-A type molecule, including one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 50, and being composed of 8 or more and 11 or less amino acid residues, is provided. All of the amino acid sequences are the amino acid sequences that are predicted to be capable of binding to an HLA-A molecule by using a prediction program utilizing an active learning method shown in FIG. 1.

## Background
TECHNICAL FIELD
The present invention relates to an HLA-binding peptide, precursor thereof, DNA fragment and recombinant vector encoding the same.
BACKGROUND ART
Human leukocyte antigen (HLA) genes are located on the short arm of chromosome 6. HLAs are classified into class I molecules (HLA-A type, B type and C type) and class II molecules (HLA-DP type, DQ type and DR type). In specific immune response, when cytotoxic T cells (CTL) recognize cell surface antigens such as tumor cells, transplanted cells and virus infected cells, it is essential to recognize the cells with class I molecules. Class I molecules are present on the membrane surface of almost all cells.
When cells are infected with a virus such as dengue virus, innate immunity is triggered to induce a reaction to eliminate the virus. After that comes specific immune response to induce a reaction to eliminate the viruses.
In specific immune response, the viruses in body fluids are eliminated by neutralizing antibodies. The viruses inside the cell are eliminated by cytotoxic T cells (CTL). Thus, CTLs specifically recognize the viral antigen (CTL epitope) composed of 8 to 11 amino acids presented by the HLA class I molecules on the infected cell surface, and eliminate the viruses by inducing damage to the infected cells. Therefore, it is important to identify such virus-specific CTL epitopes in order to produce preventive and therapeutic vaccines for the viruses.
One of such techniques is described in Patent Document 1, which discloses that an oligopeptide consisting of a certain amino acid sequence is capable of binging to HLA.

    Patent Document 1: Japanese Patent Laid-Open No. 8-151396


DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
However, there were several points to improve with the related art described in the above document, wherein the points will be described below.
First, it is not known whether the HLA-binding peptide described in the above document effectively binds to an HLA molecule. The binding to HLA could be further improved.
Second, it is described that the HLA-binding peptide described in the above document is capable of binding to HLA-DQ4. However, it is not known whether it can bind to an HLA-A2 type molecule (the product of HLA-A*0201 gene, HLA-A*0206 gene and the like) that is common in Westerners and to an HLA-A24 type molecule (the product of HLA-A*2402 gene and the like) that is common in the Japanese people.
The present invention has been made based on the above circumstances. It provides an HLA-binding peptide being excellent in binding affinity to specific types of HLA molecules.
Means for Solving the Problems
According to the present invention, there is provided an HLA-binding peptide capable of binding to an HLA-A type molecule, wherein the peptide includes one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 50, and is composed of 8 or more and 11 or less amino acid residues.
Advantage of the Invention
According to the present invention, there is provided an HLA-binding peptide being excellent in binding affinity to an HLA-A type molecule because the peptide includes a specific amino acid sequence.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic view that describes the active learning experiment design used in Examples.



BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, exemplary embodiments will be described using drawings. In all of the drawings, the same symbols are used for the same components, and descriptions will be arbitrarily abbreviated.
Exemplary Embodiment 1
In the present exemplary embodiment, peptides predicted by the hypotheses obtained by the active learning experiment method (Japanese Patent Laid-Open No. 11-316754), including an amino acid sequence whose binding affinity to an HLA molecule is defined as 3 or higher in −log Kd value, and which is composed of 8 or more and 11 or less amino acid residues were defined as candidate HLA-binding peptides. As a result of a binding experiment, the peptides were confirmed to be actually HLA-binding peptides.
As a result, a number of HLA-binding peptides were efficiently obtained, wherein the peptides were excellent binders to HLA-A type molecules because they included the amino acid sequence whose binding affinity to an HLA molecule was defined as 3 or higher in −log Kd value.
More specifically, the HLA-binding peptides in the present exemplary embodiment is an HLA-binding peptide capable of binding to an HLA-A type molecule, including one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 50 to be described below, and being composed of 8 or more and 11 or less amino acid residues.
Of the human HLA-A types, approximately 50% of the Japanese people have HLA-A24 type. Many Westerners such as Germans have HLA-A2 type.
In addition, all of the sequences are composed of 9 amino acid residues included in the certain genome protein of dengue virus, which causes dengue fever.
SEQ ID NOs: 1 to 25 are shown in Table 1 below.







TABLE 1







HLA-A24 type binding peptide











SEQ
Top 30


Binding


ID
predicted
Predicted
Sequence
experiment


NO
scores
score
name
data














1
WYAQIQPHW
6.4821
2189
8.64465


 


2
AFSGVSWTM
6.2931
727
8.19335


 


3
ILIGVVITW
6.2629
737



 


4
MMIPTVVAF
6.2445
107
7.80808


 


5
PFPQSNAPI
6.1924
1799
8.22128


 


6
LHKLGYILR
6.1857
3003



 


7
HWPKSHTLW
6.1631
999
8.65937


 


8
GLNPTAIFL
6.0286
1327
6.39195


 


9
ELPETLETL
6.0259
2140
7.00975


 


10
AWLVHRQWF
5.9475
485
8.38884


 


11
TLYAVATTF
5.9382
2282
7.58458


 


12
ALSELPETL
5.919
2137
5.505


 


13
WYMWLGARF
5.9025
2966
7.62792


 


14
ILGDTAWDF
5.8777
694
7.38687


 


15
YMPSVIEKM
5.831
2677
7.16873


 


16
KLMKITAEW
5.8074
2863
7.20712


 


17
TYGWNLVRL
5.7904
2609
7.41537


 


18
IQKETLVTF
5.7333
512
7.2378


 


19
ALHQVFGAI
5.7111
715
6.05453


 


20
GFLNEDHWF
5.6281
2981
6.7592


 


21
ALVAFLRFL
5.5909
49
5.68122


 


22
PMSTYGWNL
5.5847
2606
7.62376


 


23
AYNHALSEL
5.5579
2133
7.67924


 


24
AYTIGTTYF
5.5429
255
7.33162


 


25
DWIPLALTI
5.5177
1317
8.00347









The sequences represented by SEQ ID NOs: 1 to 25 are composed of 9 amino acid residues included in the representative strain, AY776328, of dengue virus type 2 (serotype type 2). In addition, the sequences represented by SEQ ID NOs: 1 to 25 are predicted to have high binding affinity to an HLA-A24 molecule (the product of HLA-A*2402 gene) by using the above method. SEQ ID NOs: 1 to 25 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 1 is the sequence predicted to have the highest binding affinity. Values given as predicted binding scores and experimentally obtained binding affinity data with an HLA-A24 molecule of each sequence are shown in −log Kd values.
SEQ ID NOs: 26 to 40 are shown in Table 2 below.







TABLE 2







HLA-A2 type binding peptide











SEQ
Top 30


Binding


ID
predicted
Predicted
Sequence
experiment


NO
scores
score
name
data














26
LLLTLLATV
6.3869
2149



 


27
IVLEHGSCV
5.8802
303
5.73645


 


28
LLFKTENGV
5.7903
137
7.4752


 


29
PLNEAIMAV
5.7853
1348
6.49478


 


30
NLVRLQSGV
5.5324
2613
4.69767


 


31
RLITVNPIV
5.528
630
5.64013


 


32
LLLVAHYAI
5.4766
2355
6.0465


 


33
ILIRTGLLV
5.4029
1442
6.52543


 


34
SLRPQPTEL
5.3835
878
5.0502


 


35
FLIDGPETA
5.3509
908
6.05413


 


36
FQPESPSKL
5.34
809
6.31273


 


37
ALSELPETL
5.3249
2137
6.04526


 


38
ALHQVFGAI
5.096
715
6.76833


 


39
YMPSVIEKM
5.087
2677
6.5755


 


40
ELPETLETL
5.0571
2140
6.32394









The sequences represented by SEQ ID NOs: 26 to 40 are composed of 9 amino acid residues included in the representative strain, AY776328, of dengue virus type 2. In addition, the sequences represented by SEQ ID NOs: 26 to 40 are predicted to have high binding affinity to an HLA-A2 molecule (the product of HLA-A*0201 gene) by using the above method. SEQ ID NOs: 26 to 40 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 26 is the sequence predicted to have the highest binding affinity. Values given as predicted binding scores and experimentally obtained binding affinity data with an HLA-A2 molecule of each sequence are shown in −log Kd values.
SEQ ID NOs: 41 to 50 are shown in Table 3 below.







TABLE 3







HLA-A2 type binding peptide











SEQ
Top 10


Binding


ID
predicted
Predicted
Sequence
experiment


NO
scores
score
name
data














41
LVISGLFPV
6.5075
1449
6.61148


 


42
LLLVAHYAI
6.4483
2355
6.23631


 


43
LALLAAFKV
6.2137
1202
6.46715


 


44
VILAGPMPV
6.0883
1916
6.17305


 


45
HVLGRLITV
6.0414
626
6.25386


 


46
IVLEHGSCV
6.039
303
6.02114


 


47
YVVIAILTV
5.7103
2229
5.86017


 


48
SVIEKMEAL
5.7097
2680
6.37418


 


49
RTLRVLNLV
5.6558
2651
5.96642


 


50
QILEENVEV
5.6349
2046
5.60512









The sequences represented by SEQ ID NOs: 41 to 50 are composed of 9 amino acid residues included in the representative strain, AY776328, of dengue virus type 2. In addition, the sequences represented by SEQ ID NOs: 41 to 50 are predicted to have high binding affinity to an HLA-A2 molecule (the product of HLA-A*0206 gene) by using the above method. SEQ ID NOs: 41 to 50 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 41 is the sequence predicted to have the highest binding affinity. Values given as predicted binding scores and experimentally obtained binding affinity data with an HLA-A2 molecule of each sequence are shown in −log Kd values.
As will be described in detail hereinafter, there is clearly a relationship between the predicted scores and binding experiment data in Tables 1 to 3. More specifically, the peptides predicted to have high binding affinity to an HLA-A molecule by using the above method were experimentally confirmed to have high binding affinity to an HLA-A molecule with some degree of inaccuracy.
Previously, the method for searching HLA-binding peptides by using the experiment design method has not been used. Therefore, there have been only few known HLA-binding peptides, wherein the peptides were experimentally confirmed to have binding affinity to an HLA molecule. Thus, when peptides composed of 9 amino acid residues that were randomly synthesized by the conventional method were subjected to binding experiment with an HLA-molecule, there was stochastically only 1 in approximately 100 molecules, whose binding affinity exhibited 6 or higher in −log Kd value.
In the present exemplary embodiment, the method for searching HLA-binding peptides by using the experiment design method was adopted. As a result, as many as 50 HLA-binding peptide sequences were successfully identified as described above. Moreover, some of the obtained HLA-binding peptides were experimentally tested for their binding affinity to HLA. It was confirmed that all of the tested sequences had the same or higher level of binding affinity to HLA in comparison to the prediction.
Moreover, of the sequences, an HLA-binding peptide including one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 was experimentally confirmed to have binding affinity to a human HLA-A type molecule. Therefore, the peptides are certainly regarded as HLA-binding peptides being excellent in binding affinity to a human HLA-A type molecule.
With regards to the HLA-binding peptides in the present exemplary embodiment, the binding affinity to an HLA molecule is defined as 3 or higher, particularly preferably 5 or higher, and more preferably 5.4 or higher in −log Kd value.
In the field of biochemistry, binding affinity defined as approximately 3 in −log Kd value is known to be the binding threshold for a peptide to MHCs such as HLAs. Therefore, if the binding affinity to an HLA molecule is defined as 3 or higher in −log Kd value, the peptide can be regarded as an HLA-binding peptide.
Moreover, in case of an HLA-A24 molecule, if the binding affinity to an HLA-A24 molecule is defined as 5 or higher in −log Kd value, a peptide being excellent in binding affinity to an HLA molecule can be obtained. The peptide can be preferably used to develop an effective therapeutic drug, preventive drug and the like for immune disease and the like.
In addition, if the binding affinity to an HLA-A24 molecule is defined as 5.4 or higher in −log Kd value, a peptide being particularly excellent in binding affinity to an HLA molecule can be obtained. The peptide can be preferably used to develop a further effective therapeutic drug, preventive drug and the like for immune disease and the like.
Furthermore, the HLA-binding peptide in the present exemplary embodiment may be composed of 8 or more and 11 or less amino acid residues.
If a peptide is composed of 8 or more and 11 or less amino acid residues as described above, the peptide is excellent in binding affinity to an HLA molecule. Moreover, cytotoxic T cells (CTL) specifically recognize the viral antigen (CTL epitope) composed of 8 to 11 amino acids presented by the HLA class I molecules on the surface of virus-infected cells and the like, and eliminate the viruses by inducing damage to the infected cells. Therefore, it is important to produce such virus-specific CTL epitopes composed of 8 to 11 amino acids in order to produce preventive and therapeutic vaccines for the viruses.
For example, the above HLA-binding peptide may be a peptide solely composed of amino acid residues, but it is not particularly limited thereto. For example, it may be an HLA-binding peptide precursor, wherein the precursor is modified with a sugar chain, fatty acid residue and the like according to need as long as the modification does not interfere with the effective activity of the present invention. The precursor is converted into an HLA-binding peptide by digestion with a digestive enzyme and the like in vivo in mammals such as human digestive organs. The above HLA-binding peptide exerts a similar effective activity to that of the binding peptide.
In addition, the above HLA-binding peptide may be a peptide capable of binding to a human HLA-A24 molecule.
Moreover, the above HLA-binding peptide may be a peptide capable of binding to a human HLA-A2 molecule.
According to the constitutions, a peptide that binds to an HLA-A24 molecule, which is common in Asians including the Japanese people, is obtained. Therefore, the peptide can be used to develop an effective therapeutic drug, preventive drug and the like particularly suited for Asians including the Japanese people.
Moreover, according to the constitutions, a peptide that binds to an HLA-A2 molecule, which is common in Westerners in addition to the Japanese people, is obtained. Therefore, the peptide can be used to develop an effective therapeutic drug, preventive drug and the like particularly suited for Westerners in addition to the Japanese people.
In addition, the amino acid sequence included in the above HLA-binding peptide may be an amino acid sequence derived from a certain genome protein of dengue virus, but it is not particularly limited thereto. For example, it may be an amino acid sequence derived from HIV protein, amino acid sequence derived from cedar pollen protein and the like. Moreover, it may include an amino acid sequence derived from a protein that has other pathogenicity or allergenicity.
For example, if it includes an amino acid sequence derived from dengue virus, an HLA-binding peptide that can be used to prevent and treat the disease caused by dengue virus is obtained.
Exemplary Embodiment 2
According to the present exemplary embodiment, there is provided an HLA-binding peptide capable of binding to an HLA-A type molecule, wherein the peptide comprises an amino acid sequence in which one or two amino acid residues have been deleted, substituted or added in the amino acid sequence contained in the above HLA-binding peptide, and the peptide is composed of 8 or more and 11 or less amino acid residues.
As will be described in detail below, the constitution including an amino acid sequence in which one or several amino acid residues have been deleted, substituted or added in the specific amino acid sequence that has binding affinity to an HLA-A type molecule exerts a similar effect to that of the HLA-binding peptide in the above exemplary embodiment 1.
Moreover, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule represented by SEQ ID NOs: 1 to 50, an amino acid sequence in which one or two amino acid residues have been substituted, deleted or added is predicted to exert similarly excellent binding affinity to HLA.
From another perspective, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule predicted by the above method, an amino acid sequence in which one or several amino acid residues have been substituted, deleted or added is predicted to exert similarly excellent binding affinity to HLA. Moreover, it is preferable that both the amino acid residues involved in substitution have similar properties such as hydrophobic amino acid residues.
In addition, the HLA-binding peptides described in exemplary embodiments 1 and 2 can be manufactured by a method known to a person skilled in the art. For example, the peptides can be artificially synthesized by the solid phase method or the liquid phase method. Moreover, the HLA-binding peptides can be manufactured by expressing DNA fragments or recombinant vectors encoding the HLA-binding peptides. The obtained HLA-binding peptides can be identified by a method known to a person skilled in the art. For example, they can be identified by using the Edman degradation method, mass spectrometry and the like.
Exemplary Embodiment 3
According to the present exemplary embodiment, there is provided a DNA fragment including a DNA sequence that encodes the above HLA-binding peptide. The DNA fragment in the present exemplary embodiment can express the above HLA-binding peptide because it includes the specific DNA sequence.
In addition, in order to express the above HLA-binding peptide by using the DNA fragment in the present exemplary embodiment, the DNA fragment may be introduced into the cell for expression, or may be expressed by using a commercially available artificial protein expression kit.
Moreover, the above DNA fragment may be introduced into the human cell for persistent expression. Therefore, the HLA-binding peptide is allowed to be present in the cell more persistently when the DNA fragment encoding the HLA-binding peptide is introduced into the cell than it is when the HLA-binding peptide itself is introduced into the cell. When the HLA-binding peptide is used as vaccine, it is advantageous to have the ability of persistent expression in order to enhance vaccine effectiveness.
Furthermore, the DNA fragment in the present exemplary embodiment can be manufactured by a method known to a person skilled in the art. For example, it may be artificially synthesized by a commercially available DNA synthesizer and the like. Alternatively, it may be cut out from the dengue virus genome with a restriction enzyme and the like. It can be also obtained by amplifying the dengue virus genome by the PCR method with primers. The obtained DNA fragments can be identified by a method known to a person skilled in the art. For example, they can be identified by a commercially available DNA sequencer and the like.
Exemplary Embodiment 4
According to the present exemplary embodiment, there is provided a recombinant vector including a DNA sequence that encodes the above HLA-binding peptide. The recombinant vector in the present exemplary embodiment can express the above HLA-binding peptide because it includes the specific DNA sequence.
In addition, in order to express the above HLA-binding peptide by using the recombinant vector in the present exemplary embodiment, the recombinant vector may be introduced into the cell for expression, or may be expressed by using a commercially available artificial protein expression kit.
Moreover, the above recombinant vector may be introduced into the human cell for persistent expression. Therefore, the HLA-binding peptide is allowed to be present in the cell more persistently when the recombinant vector encoding the HLA-binding peptide is introduced into the cell than it is when the HLA-binding peptide itself is introduced into the cell. When the HLA-binding peptide is used as vaccine, it is advantageous to have the ability of persistent expression in order to enhance vaccine effectiveness.
In addition, when the above recombinant vector is used, the expression level of the HLA-binding peptide can be accurately controlled by using an arbitrary sequence in the regulatory region for transcription and expression such as the promoter region upstream of the DNA sequence that encodes the above HLA-binding peptide. The number of copies of the recombinant vector in the cell can be accurately controlled by using an arbitrary sequence in the regulatory region for replication such as the origin region of the recombinant vector.
Moreover, the above recombinant vector may include an arbitrary sequence in addition to the DNA sequence encoding the above HLA-binding peptide. For example, it may include a marker gene sequence such as a drug resistance gene.
In addition, the recombinant vector in the present exemplary embodiment can be manufactured by a method known to a person skilled in the art. For example, it may be obtained by cleaving the multi-cloning site of a commercially available vector such as pBR322 and pUC19 at an arbitrary restriction site, inserting the above DNA fragment to the site, and ligating to the vector. The obtained recombinant vector can be identified by a method known to a person skilled in the art. For example, it can be identified by performing agarose gel electrophoresis to confirm whether the lengths of the cleaved DNA fragments with an arbitrary restriction enzyme match to the cleavage map of the commercially available vector such as pBR322 and pUC19, and confirming the presence of the above DNA sequence in the DNA sequence cut out from the multi-cloning site by using DNA sequencer and the like.
The above exemplary embodiments, however, are merely for exemplification of the present invention only, and various other constitutions can be adopted other than the ones described above.
For example, in the above exemplary embodiments, an HLA-binding peptide including an amino acid sequence derived from the certain genome protein of dengue virus was used. However, it may be an HLA-binding peptide including an amino acid sequence derived from another protein of dengue virus. In such cases, the peptide can be used to treat various immune diseases involving the protein from which it was derived.
Moreover, it may be an HLA-binding peptide for pathogens other than dengue virus, such as HIV virus. It may also be an HLA-binding peptide including an amino acid sequence derived from allergens such as cedar pollen, or proteins in cancer cells and the like.
In addition, when such peptide are used, if the peptides include an amino acid sequence predicted to be excellent in binding affinity to an HLA molecule by using the above method, it is considered that the peptides will be experimentally confirmed to have similarly excellent binding affinity to HLA. Therefore, the HLA-binding peptides can be preferably used for treating or preventing especially infectious diseases (dengue fever, influenza, SARS, HIV, HCV and the like) as well as cancer immunotherapy (Wilms Tumor 1, MAGE and the like), allergic disorders (pollen allergy, rheumatism, atopy, asthma and the like), autoimmune disease and the like.
EXAMPLES
Although examples will be described in detail hereinafter, it is to be understood that the invention is not restricted to these particular examples.
More specifically, the procedures for prediction, experiment and evaluation used in the present example were based on the active learning experiment design. Overall, the following steps were repeated. FIG. 1 is a schematic view that describes the active learning experiment design used herein.
(1) Run the hypostasis learning algorithm to be described hereinafter once. In other words, derive multiple hypotheses from the accumulated data by random re-sampling, and select the point that has the largest variance in the predicted values for the randomly expressed candidate query points (peptides) as the query point to be experimented.
(2) Synthesize the peptide at the selected query point by the synthesis and purification methods to be described hereinafter. Measure the actual binding affinity by the experiment to be described hereinafter, and add the affinity value to the accumulated data.
In the present example, a supervised learning algorithm based on the Hidden Markov Model was used as the hypostasis learning algorithm. Starting from the initial data for 223 peptides, a range of 20 to 30 kinds of peptides were predicted and selected per experiment. By repeating the above procedures 4 times, a total of 341 data was obtained.
More specifically, in the active learning method in the present example, we designed and synthesized a range of 20 to 30 kinds of peptides composed of an amino acid sequence that was aligned by 9 amino acids selected from the 20 amino acids in one experiment. Subsequently, we measured the strength of their binding to an HLA molecule (binding affinity). As a result, we obtained the binding affinity (Kd value) as the experimental results. The peptides with high binding affinity were regarded as the candidates for an HLA-binding peptide applicable for vaccine material.
Subsequently, we input the obtained results into the learning system equipped with learning machines that utilized the Hidden Markov Model as a mathematical algorithm in order to set the rules. We allowed each of the learning machines to take samples of different results, and set the rules. Moreover, the expressed rules were of different constitutions for each of the learning machines. In addition, the obtained rules and experimental data were loaded as the accumulated data on an as-needed basis.
Subsequently, according to the rules, we selected the next experimental candidates from the 209=500 billion peptide sequences or more, and repeated the above processes. In doing this, we applied different rules for the experimental candidates, and performed the experiment only for the candidates over which the predictions for the experimental results were divided. We could improve the final prediction accuracy because the candidates over which the predictions for the experimental results were divided were subjected to the next experiment as described above.
As described above, we performed selective sampling to select the samples that were predicted differently by multiple learning machines as the experimental candidates. As a result, we obtained information efficiently and gained the highly accurate hypotheses (rules). By repeating the above processes 4 times, excellent results in the example to be described hereinafter were obtained. Moreover, repeating the above processes 7 times or more, further excellent results can be obtained.
By performing the active learning method, we could reduce the number of binding experiments required for the peptides composed of 9 amino acid residues. In theory, all of the 500 billion candidate HLA-binding peptides or more had to be examined. In the active learning method, we prepared the rules from the experiments, and repeated the experiments for a few dozen of candidate sequences predicted by applying the rules. As a result, we could significantly reduce the time and cost required for the initial screening by cutting the number of experiments.
In addition, the frequency of encountering the HLA-binding peptides reached a range of 70 to 80% by using the prediction which uses the rules obtained by the active learning method described above. In contrast, the frequency of encountering the HLA-binding peptides by using other known technologies such as the Anchor method is merely in the range of 30%.
<Synthesis and Purification of Peptides>
Peptides were manually synthesized by using Fmoc amino acids and the Merrifield solid phase method. After deprotection, the peptides were purified by reversed phase HPLC using C18 columns to achieve the purity of 95% or more. To identify the peptides and confirm their purity, MALDI-TOF mass analysis was performed (Voyager DE RP, PerSeptive). To quantify the peptides, Micro BCA assay (Thermo Scientific Pierce) was performed by using BSA as a standard protein.
<Binding Experiment of the Peptides to an HLA-A2402 Molecule>
The peptides' binding affinity to an HLA-A24 molecule (the product of HLA-A*2402 gene) was measured by using C1R-A24 cells that express the HLA-A*2402 gene (produced by and provided with permission of Professor Masafumi Takiguchi, Kumamoto University).
First, the C1R-A24 cells were exposed to the acidic condition of pH 3.3 for 30 seconds to dissociate and remove the endogenous peptides that were originally bound to an HLA-A2402 molecule and the light chain β 2 m that was commonly assembled with HLA class I molecules. After neutralization, the C1R-A24 cells were added with purified β 2 m, and the mixture was added to a peptide serial dilution. Then, the mixture was incubated on ice for 4 hours. The 3-molecule assembly (MHC-pep) composed of the HLA-A2402 molecule, peptide and β 2 m, which had been reassembled during the incubation, was stained by using the fluorescent labeled monoclonal antibody, 17A12 that recognizes the assembly.
Subsequently, the number of MHC-pep per C1R-A24 cell (which is proportional to the fluorescent intensity of the above fluorescent antibody) was quantitatively measured by using a fluorescent cell analysis machine, FACScan (Becton, Dickinson and Company). The binding dissociation constant, Kd value, between the HLA-A24 molecule and peptide was calculated from the average fluorescent intensity per cell by using the method published in the research article (Udaka et al., Immunogenetics, 51, 816-828, 2000).
<Binding Experiment of the Peptides to an HLA-A0201 Molecule>
The peptides' binding affinity to an HLA-A2 molecule (the product of HLA-A*0201 gene) was measured by using the cell line JY that expresses the HLA-A*0201 gene (obtained from ATCC (American Type Culture Collection)).
First, the JY cells were exposed to the acidic condition of pH 3.8 for 30 seconds to dissociate and remove the endogenous peptides and light chain β 2 m that had been non-covalently bound to an HLA-A0201 molecule. After neutralization, a reassemble experiment was performed.
The above JY cells and purified β 2 m were added to a serial dilution of the peptide whose binding affinity was to be measured. Then, the mixture was incubated on ice for 4 hours. The HLA-A0201 molecule that had been reassembled up to this point was stained by using the assembly-specific fluorescent labeled monoclonal antibody, BB7.2.
Subsequently, the amount of fluorescence per cell was measured by using a flow cytometer. The dissociation constant, Kd value, was calculated by using the method published in the research article (Udaka et al., Immunogenetics, 51, 816-828, 2000).
<Binding Experiment of the Peptides to an HLA-A0206 Molecule>
The peptides' binding affinity to an HLA-A2 molecule (the product of HLA-A*0206 gene) was measured by using RA2.6 cells (a newly prepared cell line at Kochi University) that express the HLA-A*0206 gene, which were created by expressing cDNA of the HLA-A*0206 gene in RAMS cells, the mouse TAP peptide transporter deficient cells.
First, the RA2.6 cells were cultured overnight at 26° C. to accumulate HLA-A0206 molecules that were unbound to peptides on the cell surface. A peptide serial dilution was added thereto for binding at room temperature for 30 minutes.
Subsequently, the mixture was cultured for 3.5 hours at 37° C. to denature the empty HLA-A0206 molecules that were unbound to peptides. As a result, the HLA-A0206 molecules lost their steric structure.
Either the fluorescent labeled monoclonal antibody 17A10 or 17A12, which specifically recognizes a peptide-bound HLA-A0206 molecule, was added thereto. The mixture was incubated on ice for 20 minutes, and the cells were stained.
Subsequently, the amount of fluorescence per cell was measured by using a flow cytometer. The dissociation constant, Kd value, was calculated by using the method published in the research article (Udaka et al., Immunogenetics, 51, 816-828, 2000).
<Evaluation Results>
As a result, the prediction results and experimental results shown in the above Tables 1 to 3 were obtained.
The sequences represented by SEQ ID NOs: 1 to 25 in Table 1 are the sequences composed of 9 amino acid residues included in the representative strain, AY776328, of dengue virus type 2 that is registered in GENBANK. Moreover, the sequences represented by SEQ ID NOs: 1 to 25 are the sequences which were predicted to have high binding affinity to an HLA-A24 molecule (the product of HLA-A*2402 gene) using the hypotheses obtained by the experiment design method described in the exemplary embodiment 1. In addition, SEQ ID NOs: 1 to 25 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 1 is the sequence predicted to have the highest binding affinity. Moreover, the entire amino acid sequence of the representative strain, AY776328, of dengue virus type 2







(MNNQRKKARNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMA


 


LVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRR


 


RTAGVIIMMIPTVVAFHLTTRNGEPHMIVSRQEKGKSLLFKTENGVNMCT


 


LMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYGTCTATGE


 


HRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVLRHPGFTI


 


MAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFVEGVSGGS


 


WVDIVLEHGSCVTTMAKNKPTLDFELTKTEAKHPATLRKYCIEAKLTNTT


 


TASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMF


 


TCKKNMEGKVVQPENLEYTIVITPHSGEENAVGNDTGKHGKEIKVTPQSS


 


ITEAELTGYGTVTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLPLP


 


WLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATE


 


IQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGT


 


IVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEA


 


EPPFGDSYIIIGVEPGQLKLSWFKKGSSIGQMFETTMRGAKRMAILGDTA


 


WDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVVITWIGMN


 


SRSTSLSVSLVLVGVVTLYLGVMVQADSGCVVSWKNKELKCGSGIFITDN


 


VHTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPE


 


LNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSWKAWGKAKML


 


STELHNHTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLKER


 


QDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKSCH


 


WPKSHTLWSNGVLESEMIIPKNFAGPVSQHNYRPGYHTQTAGPWHLGKLE


 


MDFDFCEGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYR


 


GEDGCWYGMEIRPLKEKEENLVNSLVTAGHGQIDNFSLGVWGMALFLEEM


 


LRTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGV


 


TYLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILE


 


LTDALALGMMVLKIVRNMEKYQLAVTIMAILCVPNAVILQNAWKVSCTIL


 


AVVSVSPLLLTSSQQKADWIPLALTIKGLNPTAIFLTTLSRTSKKRSWPL


 


NEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELE


 


RAADVRWEEQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLL


 


VISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPVGKAELEDGAYR


 


IKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKK


 


DLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNTGTIG


 


AVSLDFSPGTSGSPIVDKKGKVVGLYGNGVVTRSGTYVSAIAQTEKSIED


 


NPEIEDDIFRKKRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPT


 


RVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPIR


 


VPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDP


 


FPQSNAPIMDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAAC


 


LRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVID


 


PRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRVGRNPKNENDQYIY


 


MGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYR


 


LRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILE


 


ENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGRKSLTLNL


 


ITEMGRLPTFMTQKARNALDNLAVLHTAEAGGRAYNHALSELPETLETLL


 


LLTLLATVTGGIFLFLMSGKGIGKMTLGMCCIITASVLLWYAQIQPHWIA


 


ASIILEFFLIVLLIPEPEKQRTPQDNQLTYVVIAILTVVAATMANEMGFL


 


EKTKKDFGLGGIATQQPESNILDIDLRPASAWTLYAVATTFITPMLRHSI


 


ENSSVNVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNPIT


 


LTAALLLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVIDLD


 


PIPYDPKFEKQLGQVMLLVLCWQVLMMRTTWALCEALTLATGPISTLWEG


 


NPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTANTRRGTGNTGETL


 


GEKWKNRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGETDHHAVSRGS


 


AKLRWFVERNLVTPEGKVMDLGCGRGGWSYYCGGLKNVKEVKGLTKGGPG


 


HEEPIPMSTYGWNLVRLQSGVDVFFTPPEKCDTLLCDIGESSPNPTVEAG


 


RTLRVLNLVENWLNNNTQFCIKVLNPYMPSVIEKMEALQRKYGGALVRNP


 


LSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRHKKATYEPDVDLG


 


SGTRNIGIESEIPNLDIIGKRIEKIKQEHETSWHYDQDHPYKTWAYHGSY


 


ETKQTGSASSMVNGVVRLLTKPWDVIPMVTQMAMTDTTPFGQQRVFKEKV


 


DTRTQEPKEGTKKLMKITAEWLWKELGKKKTPRMCTREEFTRKVRSNAAL


 


GAIFTDENKWKSAREAVEDSGFWELVDKERNLHLEGKCETCVYNMMGKRE


 


KKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSLSGVER


 


EGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNHMEG


 


EHKKLAEAIFKLTYQNKVVRVQRPTPRGTVMDIISRRDQRGSGQVVTYGL


 


NTFTNMEAQLIRQMEGEGVFKSIQQLTATEEIAVKNWLARVGRERLSRMA


 


ISGDDCVVKPLDDRFASALTALNDMGKVRKDIQQWEPSRGWNDWTQVPFC


 


SHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWSLRETACLGKSYA


 


QMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHATHEWMTTEDM


 


LTVWNRVWIQENPWIEDKTPVESWEEIPYLGKREDQWCGSLIGLTSRATW


 


AKNIQTAINQVRSLIGNEEYTDYMPSMKRFRREEEEAGVLW)






is shown in SEQ ID NO: 51

In addition, the sequences represented by SEQ ID NOs: 26 to 40 in Table 2 are the sequences composed of 9 amino acid residues included in the representative strain, AY776328, of the above dengue virus type 2. Moreover, the sequences represented by SEQ ID NOs: 26 to 40 are the sequences which were predicted to have high binding affinity to an HLA-A2 molecule (the product of HLA-A*0201 gene) using the hypotheses obtained by the experiment design method described in the exemplary embodiment 1. In addition, SEQ ID NOs: 26 to 40 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 26 is the sequence predicted to have the highest binding affinity.
Furthermore, the sequences represented by SEQ ID NOs: 41 to 50 in Table 3 are the sequences composed of 9 amino acid residues included in the representative strain, AY776328, of the above dengue virus type 2. Moreover, the sequences represented by SEQ ID NOs: 41 to 50 are the sequences which were predicted to have high binding affinity to an HLA-A2 molecule (the product of HLA-A*0206 gene) using the hypotheses obtained by the experiment design method described in the exemplary embodiment 1. In addition, SEQ ID NOs: 41 to 50 are listed in the descending order of binding affinity. More specifically, SEQ ID NO: 41 is the sequence predicted to have the highest binding affinity.
In addition, Tables 1 to 3 show the amino acid sequences corresponding to the top predicted score results by using the above prediction program, predicted scores, and the corresponding binding experiment data with regards to the 9 amino acid residues included in the representative strain, AY776328, of the above dengue virus type 2. Moreover, all of the binding experiments were performed by artificially synthesizing the 9 amino acid peptides by the above synthesis method.
All of the above peptide sequences in which one or two amino acid residues are substituted respectively are predicted to exert similarly excellent binding affinity to an HLA-A molecule. Thus, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule represented by SEQ ID NOs: 1 to 50, an amino acid sequence in which one or several amino acid residues have been substituted, deleted or added is predicted to exert similarly excellent binding affinity to HLA.
From another perspective, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule, wherein the sequences were predicted by the hypotheses obtained by the experiment design method described in the exemplary embodiment 1, an amino acid sequence in which one or several amino acid residues have been substituted, deleted or added is regarded to exert similarly excellent binding affinity to HLA. Moreover, it is preferable that the amino acid residues to be substituted respectively have similar properties such as hydrophobic amino acid residues.
One of the present inventors, Udaka, et al. has already reported that the peptide sequences in which one or two amino acid residues have been substituted respectively show similarly excellent binding affinity to an antigen presenting molecule.

    1. “Decrypting the structure of MHC-I restricted CTL epitopes with complex peptide libraries.” Keiko Udaka, Karl-Heinz Wiesmuller, Stefan Kienle, Gunter Jung and Peter Walden. J. Exp. Med. 181, 2097-2108. (1995)
    2. “Tolerance to amino acid varidations in peptides binding to the MHC class I protein H-2Kb.” Keiko Udaka, Karl-Heinz Wiesmuller, Stefan Kienle, Gunter Jung and Peter Walden. J. Biol. Chem. 270, 24130-24134. (1995)
    3. “Self MHC-restricted peptides recognized by all alloreactive T lymphocyte clone.” Keiko Udaka, Karl-Heinz Wiesmuller, Stefan Kienle, Gunter Jung and Peter Walden. J. Immunol. 157, 670-678. (1996)


Therefore, the above peptide sequences in which one or two amino acid residues have been substituted respectively are predicted to exert similarly excellent binding affinity to an HLA-A molecule. Thus, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule represented by SEQ ID NOs: 1 to 50, an amino acid sequence in which one or several amino acids have been substituted, deleted or added is predicted to exert similarly excellent binding affinity to HLA.
From another perspective, of the amino acid sequences being excellent in binding affinity to an HLA-A molecule, wherein the sequences were predicted by the hypotheses obtained by the experiment design method described in the exemplary embodiment 1, an amino acid sequence in which one or several amino acid residues have been substituted, deleted or added is regarded to exert similarly excellent binding affinity to HLA. Moreover, it is preferable that the amino acid residues to be substituted respectively have similar properties such as hydrophobic amino acid residues.
The present invention has been described based on the examples. However, the examples are merely for exemplification only. It is to be understood by those skilled in the art that various modifications can be made and are within the scope of this invention.
This application claims priority based on Japanese Patent Application No: 2006-279232 filed on Oct. 12, 2006 and Japanese Patent Application No: 2007-189047 filed on Jul. 20, 2007. The disclosure of the applications is incorporated herein in its entirety.

## Claims

1. An HLA-binding peptide capable of binding to an HLA-A type molecule, characterized in that the peptide comprises the amino acid sequence of SEQ ID NO: 15, and wherein said HLA-binding peptide is composed of 11 or less amino acid residues.

2. An HLA-binding peptide capable of binding to an HLA-A type molecule, characterized in that the peptide comprises the amino acid sequence of SEQ ID NO: 15 in which one amino acid residue has been deleted, substituted or added and wherein said HLA-binding peptide is composed of 8 or more and 11 or less amino acid residues.

3. The HLA-binding peptide according to claim 1, characterized in that the HLA-binding peptide binds to an HLA-A24 molecule.

4. The HLA-binding peptide according to claim 3, wherein the HLA-A24 molecule is an HLA-A2402 molecule.

5. The HLA-binding peptide according to claim 1, characterized in that the HLA-binding peptide binds to an HLA-A2 molecule.

6. The HLA-binding peptide according to claim 5, wherein the HLA-A2 molecule is an HLA-A0201 molecule.

7. The HLA-binding peptide according to claim 5, wherein the HLA-A2 molecule is an HLA-A0206 molecule.