Antigenic Peptides For Prevention And Treatment Of B-Cell Malignancy

Abstract

The present invention relates to antigen-based immunotherapy, in particular cancer immunotherapy. In particular, the present invention provides antigenic peptides, which are distinct from, but have amino acid similarity to, especially share the same core sequence with epitopes of human tumor antigens. The present invention further provides immunogenic compounds, nanoparticles, cells and pharmaceutical compositions comprising such antigenic peptides and nucleic acids encoding such antigenic peptides.

Claims

1.-5. (canceled)

6. An antigenic peptide comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs 1-257 and 476-500, wherein zero, 1 or 2 amino acid residues are substituted, deleted or added.

7. The antigenic peptide according to claim 6, wherein the core sequence is maintained.

8.-10. (canceled)

11. The antigenic peptide according to claim 6, wherein the antigenic peptide has a length of 9 or 10 amino acids.

12. The antigenic peptide according to claim 6, wherein the antigenic peptide binds to MHC class I molecules.

13. The antigenic peptide according to claim 6, wherein the antigenic peptide induces T-cell cross-reactivity against a reference epitope of a human B-cell tumor antigen, which shares the same core sequence.

14. The antigenic peptide according to claim 6, wherein the antigenic peptide comprises a (core) sequence according to any one of SEQ ID NOs 281-303.

15. The antigenic peptide according to claim 6, wherein a reference epitope of a human B-cell tumor antigen, which shares the same core sequence, binds weaker to MHC I molecules than the antigenic peptide.

16. The antigenic peptide according to claim 6, wherein the antigenic peptide is a microbiota variant identified in at least one protein expressed in a human microbiota.

17. (canceled)

18. The antigenic peptide according to claim 6, wherein the antigenic peptide has a cleavage probability score higher than 70%.

19.-22. (canceled)

23. The antigenic peptide according to claim 6, wherein the antigenic peptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 110.

24. The antigenic peptide according to claim 6, wherein the antigenic peptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 114.

25. The antigenic peptide according to claim 6, wherein the antigenic peptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 220.

26. The antigenic peptide according to claim 6, wherein the antigenic peptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 65.

27.-30. (canceled)

31. The antigenic peptide according to claim 6, wherein the length of the antigenic peptide does not exceed 10, 15, 20, 25 or 30 amino acids.

32. (canceled)

33. An immunogenic compound comprising the antigenic peptide according to claim 6.

34.-37. (canceled)

38. A cell loaded with the antigenic peptide according to claim 6 or with an immunogenic compound comprising the antigenic peptide.

39. (canceled)

40. A nucleic acid encoding an antigenic peptide comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs 1-257 and 476-500, wherein zero, 1 or 2 amino acid residues are substituted, deleted or added; or a polypeptide comprising the antigenic peptide.

41. (canceled)

42. A host cell comprising the nucleic acid according to claim 40, wherein the nucleic acid is a vector.

43. (canceled)

44. (canceled)

45. A cytotoxic T lymphocyte (CTL) specific for an antigenic peptide according to claim 6.

46. A pharmaceutical composition comprising an antigenic peptide comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs 1-257 and 476-500, wherein zero, 1 or 2 amino acid residues are substituted, deleted or added, an immunogenic compound comprising the antigenic peptide, a nanoparticle comprising the antigenic peptide, a cell comprising the antigenic peptide, a nucleic acid encoding the antigenic peptide, a host cell comprising the antigenic peptide, or a cytotoxic T lymphocyte (CTL) specific for the antigenic peptide, and, optionally, one or more pharmaceutically acceptable excipients or carriers.

47. The pharmaceutical composition according to claim 46, wherein the composition comprises (i) at least two distinct antigenic peptides; (ii) at least two distinct immunogenic compounds; (iii) at least two distinct nanoparticles; (iv) at least two distinct nucleic acids; or (v) at least two distinct cytotoxic T lymphocytes.

48. The pharmaceutical composition according to claim 47 comprising at least three or four distinct components according to any one of (i)-(v).

49. The pharmaceutical composition according to claim 48, wherein the at least three or four distinct components relate to the antigenic peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 110; the antigenic peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 114; the antigenic peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 220; and optionally, the antigenic peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 65.

50. The pharmaceutical composition according to claim 46 further comprising a helper peptide.

51. A kit comprising the antigenic peptide according to claim 6, or a pharmaceutical composition comprising the antigenic peptide.

52. (canceled)

53. The kit according to claim 51, wherein the kit comprises at least two distinct antigenic peptides.

54.-57. (canceled)

58. A combination of at least two distinct antigenic peptides according to claim 6.

59.-72. (canceled)

73. A method for preventing and/or treating a B-cell malignancy or initiating, enhancing or prolonging an anti-tumor-response against a B-cell malignancy in a subject in need thereof, the method comprising administering to the subject an antigenic peptide comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs 1-257 and 476-500, wherein zero, 1 or 2 amino acid residues are substituted, deleted or added, an immunogenic compound comprising the antigenic peptide, a nanoparticle comprising the antigenic peptide, a cell comprising the antigenic peptide, a nucleic acid encoding the antigenic peptide, a host cell comprising the antigenic peptide, a cytotoxic T lymphocyte specific for the antigenic peptide, a pharmaceutical composition comprising the antigenic peptide, or a combination comprising the antigenic peptide and at least one other antigenic peptide.

74. The method according to claim 73, wherein the a B-cell malignancy is selected from a B-cell lymphoma selected from the group consisting of non-Hodgkin lymphoma (NHL), diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt'-s lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma (FL).

75. A peptide-MHC (pMHC) multimer comprising the antigenic peptide according to claim 6.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0442] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0443] FIG. 1: shows for Example 1 in vitro affinity for the antigenic peptide CD22-B1 in comparison to the corresponding human CD22 epitope CD22-H1.

[0444] FIG. 2: shows for Example 1 in vitro affinity for the antigenic peptide CD37-B1 in comparison to the corresponding human CD37 epitope CD37-H1.

[0445] FIG. 3: shows for Example 1 in vitro affinity for the antigenic peptide CD19-B1 in comparison to the corresponding human CD19 epitope CD19-H1.

[0446] FIG. 4: shows for Example 1 in vitro affinity for the antigenic peptide CD19-B2 in comparison to the corresponding human CD19 epitope CD19-H2.

[0447] FIG. 5: shows for Example 1 in vitro affinity for the antigenic peptide TNFRSF13C-B1 in comparison to the corresponding human TNFRSF13C epitope TNFRSF13C-H1.

[0448] FIG. 6: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide CD22-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide CD22-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0449] FIG. 7: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide TNFRSF13C-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide TNFRSF13C-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0450] FIG. 8: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide CD37-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide CD37-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0451] FIG. 9: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide CD19-B2 as indicated in the figure and cross-reactivity with the human corresponding peptide CD19-H2. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0452] FIG. 10: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide CD19-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide CD19-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0453] FIG. 11: shows for Example 2 ELISPOT results for HHD DRs HLA-A2 transgenic mice vaccinated with the antigenic peptide CD22-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide CD22-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0454] FIG. 12: shows for Example 2 ELISPOT results for HHD DRs HLA-A2 transgenic mice vaccinated with the antigenic peptide TNFRSF13C-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide TNFRSF13C-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0455] FIG. 13: shows for Example 2 ELISPOT results for HHD DRs HLA-A2 transgenic mice vaccinated with the antigenic peptide CD37-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide CD37-H1. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0456] FIG. 14: shows for Example 1 in vitro affinity for the antigenic peptide MS4A1-B4 in comparison to the corresponding human MS4A1 epitope MS4A1-H4.

[0457] FIG. 15: shows for Example 2 ELISPOT results for HHD DR1 HLA-A2 transgenic mice vaccinated with the antigenic peptide MS4A1-B4 as indicated in the figure and cross-reactivity with the human corresponding peptide MS4A1-H4. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0458] FIG. 16: shows for Example 2 ELISPOT results for HHD DRs HLA-A2 transgenic mice vaccinated with the antigenic peptide MS4A1-B4 as indicated in the figure and cross-reactivity with the human corresponding peptide MS4A1-H4. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells.

[0459] FIG. 17: shows for Example 3 the detection of CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4 peptide-specific CD8+ T cells detected in peripheral blood from healthy donors (HLA-A2 positive).

[0460] FIG. 18: shows for Example 3 the cytotoxic capacity of the CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4 peptide-specific human T cells clone expanded in vitro by microbiome derived peptide stimulation. CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4 peptide-specific T cells have the ability to kill T2 cells loaded with bacterial or human peptides.

[0461] FIG. 19: shows for Example 4 in vitro affinity for the antigenic peptides CD22-B1, CD22-B12 and CD22-B13 in comparison to the corresponding human CD22 epitope CD22-H1.

[0462] FIG. 20: shows for Example 4 in vitro affinity for the antigenic peptides CD37-B1, CD37-B12, CD37-B11, CD37-B14 and CD37-B15 in comparison to the corresponding human CD37 epitope CD37-H1.

[0463] FIG. 21 shows for Example 4 in vitro affinity for the antigenic peptides TNFRSF13C-B1, TNFRSF13C-B11, TNFRSF13C-B12 and TNFRSF13C-B13 in comparison to the corresponding human TNFRSF13C epitope TNFRSF13C-H1.

[0464] FIG. 22: shows for Example 4 in vitro affinity for the antigenic peptide MS4A1-B4, MS4A1-B42 and MS4A1-B43 in comparison to the corresponding human MS4A1 epitope MS4A1-H4.

EXAMPLES

[0465] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Antigenic Peptides have Superior Affinity to the HLA-A*0201 Allele

[0466] Next, binding affinity of various selected antigenic peptides and of the corresponding fragments of human tumor antigens (human reference peptides) to the HLA-A*0201 allele was confirmed in vitro. Namely, the antigenic peptide of sequence SEQ ID NO: 110 («YIFEHPELL» also referred herein as CD22-B1) was compared to the corresponding reference human peptides derived from CD22 («WVFEHPETL», SEQ ID NO: 270, also referred herein as CD22-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 109 («YVFEHPELL» also referred herein as CD22-B11) was compared to the corresponding reference human peptide derived from CD22 («WVFEHPETL», SEQ ID NO: 270, also referred herein as CD22-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 114 («FLAFVPLQL» also referred herein as CD37-B1i) was compared to the corresponding reference human peptide derived from CD37 («GLAFVPLQI», SEQ ID NO: 271, also referred herein as CD37-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 117 («GMAFVPLLL» also referred herein as CD37-B11i) was compared to the corresponding reference human peptide derived from CD37 («GLAFVPLQI», SEQ ID NO: 271, also referred herein as CD37-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 34 («LLVGILHLV» also referred herein as CD19-B1) was compared to the corresponding reference human peptide derived from CD19 («SLVGILHLQ», SEQ ID NO: 260, also referred herein as CD19-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 10 («TLLFLTPML» also referred herein as CD19-B2) was compared to the corresponding reference human peptide derived from CD19 («FLLFLTPME», SEQ ID NO: 258, also referred herein as CD19-H2). Moreover, the antigenic peptide of sequence SEQ ID NO: 39 («YLAYLIFEL» also referred herein as CD19-B6) was compared to the corresponding reference human peptide derived from CD19 («TLAYLIFCL», SEQ ID NO: 261, also referred herein as CD19-H6). Moreover, the antigenic peptide of sequence SEQ ID NO: 40 («LQMGGFYLL» also referred herein as CD19-B7) was compared to the corresponding reference human peptide derived from CD19 («QQMGGFYLC», SEQ ID NO: 262, also referred herein as CD19-H7). Moreover, the antigenic peptide of sequence SEQ ID NO: 220 («LMFGAPALV» also referred herein as TNFRSF13C-B1) was compared to the corresponding reference human peptide derived from TNFRSF13C («LLFGAPALL», SEQ ID NO: 279, also referred herein as TNFRSF13C-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 231 («ILPGLLFGL» also referred herein as TNFRSF13C-B31) was compared to the corresponding reference human peptide derived from TNFRSF13C («PLPGLLFGA», SEQ ID NO: 280, also referred herein as TNFRSF13C-H3). Moreover, the antigenic peptide of sequence SEQ ID NO: 227 («FMPGLLFGA» also referred herein as TNFRSF13C-B33) was compared to the corresponding reference human peptide derived from TNFRSF13C («PLPGLLFGA», SEQ ID NO: 280, also referred herein as TNFRSF13C-H3). Moreover, the antigenic peptide of sequence SEQ ID NO: 61 («YILGGLLMV» also referred herein as MS4A1-B12) was compared to the corresponding reference human peptide derived from MS4A1 (also known as CD20) («IALGGLLMI», SEQ ID NO: 263, also referred herein as MS4A1-H1). Moreover, the antigenic peptide of sequence SEQ ID NO: 72 («ILIPAGIYL» also referred herein as MS4A1-B3) was compared to the corresponding reference human peptide derived from MS4A1 (also known as CD20) («LMIPAGIYA», SEQ ID NO: 265, also referred herein as MS4A1-H3). Moreover, the antigenic peptide of sequence SEQ ID NO: 65 («AMNSLSLYI» also referred herein as MS4A1-B4) was compared to the corresponding reference human peptide derived from MS4A1 (also known as CD20) («IMNSLSLFA», SEQ ID NO: 264, also referred herein as MS4A1-H4). Moreover, the antigenic peptide of sequence SEQ ID NO: 86 («YLFLGILSL» also referred herein as MS4A1-B5) was compared to the corresponding reference human peptide derived from MS4A1 (also known as CD20) («SLFLGILSV», SEQ ID NO: 266, also referred herein as MS4A1-H5).

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

[0467] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP ½ negative and incapable of presenting endogenous peptides.

[0468] T2 cells (5.104 cells per well) are incubated with decreasing concentrations of peptides from 100 μM to 0.1 μM (4 points: 100 μM, 10 μM, 1 μM, 0.1 μM) in serum-free medium (TexMacs) supplemented with 100 ng/μl of β2 Microglobulin at 37° C. for 16 hours. Cells are then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).

[0469] The analysis is achieved by FACS (Macsquant analyzer 10-Miltenyi).

[0470] For each peptide concentration, the geometric mean of the labelling associated with the peptide of interest is subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM. The relative affinity is then determined as follows:

relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.

A2. Solubilisation of Peptides

[0471] Each peptide is solubilized by taking into account the amino acid composition. For peptides which do not include any Cystein, Methionin, or Tryptophane, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are resuspended in water or PBS pH7.4.

B. Results

[0472] The mean relative fluorescence intensity values (data are normalized to the mean fluorescence of HIV peptide, i.e. a value of 100 is equal to the best binding observed with HIV peptide) of T2 cells obtained for the various concentrations of each peptide are shown in Table 2 below:

TABLE-US-00004 TABLE 2 Peptide Name SEQ ID NO. 100 10 1 0.1 CD22-B1 110 164 123 81 0 CD22-B11 109 140.7 123.8 60.5 6.4 CD22-H1 270 158 71 10 2 CD37-B1 114 157 106 35 5 CD37-B11 117 57.8 48.9 1.6 0.0 CD37-H1 271 153 27 0 2 CD19-B1 34 93 40 8 ND CD19-B11 35 108.5 30.5 5.2 0 CD19-H1 260 0 1 0 1 CD19-B2 10 ND 114 41 12 CD19-H2 258 7 7 3 ND CD19-B6 39 ND 52.5 5.4 0 CD19-H6 261 ND 39.6 4.1 0 CD19-B7 40 31.8 4.5 4.8 6 CD19-H7 262 9.3 2 0.1 0 TNFRSF13C-B1 220 90 37 7 1 TNFRSF13C-H1 279 92 17 2 0 TNFRSF13C-B31 231 104.3 71.9 1.5 0 TNFRSF13C-B33 227 99.6 15.5 1.3 0 TNFRSF13C-H3 280 8.4 0 0 0 MS4A1-B12 61 42 7.6 1.1 1.2 MS4A1-H1 263 ND 1.9 0.7 0 MS4A1-B3 72 88.8 87.8 17.8 1.9 MS4A1-H3 265 92.6 35.7 0.9 0 MS4A1-B4 65 115.2 13.1 1.9 0.5 MS4A1-H4 264 13.4 2.6 0.3 0 MS4A1-B5 86 ND 29.8 0.2 0 MS4A1-H5 266 ND 6.5 1.8 0

[0473] Table 3 below summarizes for each tested peptide the concentration required to induce 20% of HLA-A2 expression and the in vitro binding affinity (* normalized against HIV-pol concentration of peptide inducing 20% of HLA-A2 expression performed during the same experiment).

TABLE-US-00005 TABLE 3 ND-Undeterminable Concentration of peptide In vitro SEQ ID that induces 20% of binding Peptide NO HLA-A2 expression (μM) affinity* CD22-B1 110 0.16 0.4 CD22-B11 109 0.2 0.5 CD22-H1 270 1.97 5.3 CD37-B1 114 0.65 1.6 CD37-B11 117 2.30 3 CD37-H1 271 7.35 17.5 CD19-B1 34 3.7 0.7 CD19-B11 35 5.97 0.8 CD19-H1 260 ND ND CD19-B2 10 0.38 1 CD19-H2 258 ND ND CD19-B6 39 3.76 0.4 CD19-H6 261 4.98 0.5 CD19-B7 40 49.63 6.8 CD19-H7 262 >100 ND TNFRSF13C-B1 220 4.35 1.1 TNFRSF13C-H1 279 11.76 3.0 TNFRSF13C-B31 231 1.60 0.40 TNFRSF13C-B33 227 13.20 3.50 TNFRSF13C-H3 280 ND ND MS4A1-B12 61 31.20 3.50 MS4A1-H1 263 ND ND MS4A1-B3 72 0.70 0.10 MS4A1-H3 265 4.83 0.50 MS4A1-B4 65 12.3 2.1 MS4A1-B41 68 2.45 0.30 MS4A1-H4 264 >100 ND MS4A1-B5 86 6.77 0.70 MS4A1-H5 266 ND ND

[0474] In addition, FIGS. 1-5 and 14 illustrate the results for selected examples, namely for the antigenic peptide CD22-B1 in comparison to the corresponding human CD22 fragment CD22-H1 (Figure i), for the antigenic peptide CD37-B1, in comparison to the corresponding human CD37 fragment CD37-H1 (FIG. 2), for the antigenic peptide CD19-B31 in comparison to the corresponding human CD19 fragment CD19-H1 (FIG. 3), for the antigenic peptide CD19-B32 in comparison to the corresponding human CD19 fragment CD19-H2 (FIG. 4), for the antigenic peptide TNFRSF13C-B1 in comparison to the corresponding human TNFRSF13C fragment TNFRSF13C-H1 (FIG. 5) and the antigenic peptide MS4A1-B4 in comparison to the corresponding human MS4A1 fragment MS4A1-B4 (FIG. 14).

[0475] In summary, the results show that the antigenic peptides according to the present invention show at least similar binding affinity to HLA-A*0201 as the corresponding human tumor antigen fragments. In most cases, the binding affinity observed for the antigenic peptides according to the present invention was stronger than that of the corresponding human epitopes. Without being bound to any theory it is assumed that such a strong binding affinity of the antigenic peptides according to the present invention reflects their ability to raise an immune response (i.e., their immunogenicity).

Example 2: Immunogenicity of CD22-B1, CD19B1, CD19-B2, CD37B1, TNFRSF13C-B1 and MS4A1-B4 in HLA-A2 Transgenic Mice and Cross-Reactivity with the Corresponding Human Peptide

A. Materials and Methods

A.1 Mouse Model

[0476] Briefly, HLA-A2 HHD-DR1 humanized mice (C57BL/6JB2mtm1UncIAb-/-Tg(HLA-DRA, HLA-DRB1*0101)#GjhTg(HLA-A/H2-D/B2M)1Bpe) or HHD-DR3 humanized mice (C57BL/6JB2mtm1UncIAb-/-Tg(HLA-DRA,HLA-DRB1*0301)#GjhTg(HLA-A/H2-D/B2M)1Bpe) were assigned randomly (based on mouse sex and age) to experimental groups, wherein each group was immunized with a specific vaccination peptide (vacc-pAg) combined to a common helper peptide (h-pAg UCP2; sequence: KSVWSKLQSIGIRQH; SEQ ID NO: 475; for HHD DR1 mice or h-pAg DR3; sequence MAKTIAYDEEARRGLERGLN; SEQ ID n° 473; for HHD DR3 mice) (as outlined in Table 4 below).

TABLE-US-00006 TABLE 4 Experimental group composition. h-pAg: ‘helper’ peptide; vacc-pAg: vaccination peptide. The number of boost injections is indicated into brackets. Peptide Helper Animal Group (vacc-pAg) (h-Ag) Mice Prime Boost Number 1 CD22-B1 UCP2 HHD- + +(1X) 6 (60 nMole) (105 nMole) DR1 2 CD19-B1 UCP2 HHD- + +(1X) 6 (60 nMole) (105 nMole) DR1 3 CD19-B2 UCP2 HHD- + +(1X) 6 (60 nMole) (105 nMole) DR1 4 CD37-B1 UCP2 HHD- + +(1X) 5 (60 nMole) (105 nMole) DR1 5 TNFRSF13C- UCP2 HHD- + +(1X) 5 B1 (60 nMole) (105 nMole) DR1 6 CD22-B1 DR3 (65 nM) HHD- + +(1X) 5 (95 nMole) DR3 7 TNFRSF13C- DR3 (65 nM) HHD- + +(1X) 5 B1 (95 nMole) DR3 8 CD37-B1 DR3 (65 nM) HHD- + +(1X) 5 (95 nMole) DR3 9 MS4A1-B4 UCP2 HHD- + +(1X) 6 (60 nMole) (105 nMole) DR1 10 MS4A1-B4 DR3 (65 nM) HHD- + +(1X) 5 95 nMole DR3

[0477] The peptides were provided as follows: [0478] vacc-pAg: CD22-B1, CD19-B1, CD19-B2, CD37-B1, TNFRSF13C-B1 and MS4A1-B4 all produced and provided at a 4 mg/ml (4 mM) concentration; [0479] h-pAg: DR3 or UCP2 re-suspended in pure distilled water at a 10 mg/mL concentration

[0480] The peptide formulation to be injected (emulsion) was prepared freshly on each day of injection and for each group. A mix for 10 animals was prepared using 2 mL luer lock syringes (460670 IV, B BRAUN) and luer connectors (Cole-Parmer, 45502-22): 500 μL of peptide mixture in syringe 1 was emulsified with 500 μL of IFA contained in syringe 2, at high speed as fast as possible until forming a thick (white foam) emulsion. Each emulsion was prepared in excess to compensate for the dead volumes at injection.

[0481] The animals were immunized on day 0 (do) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 μL of an oil-based emulsion that contained: [0482] 60nMole of vacc-pAg; 105nMole of UCP2 helper peptide (for HHD-DR1 mice) or 65nMole of DR3 helper peptide (for HHD-DR3 mice) [0483] 10 μL of PBS to reach a total volume of 50 μL (per mouse); [0484] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 μL per mouse).

A.2 Analysis

[0485] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 μm-filtering and Ficoll density gradient purification.

[0486] The cell suspensions were further used in an ELISPOT-IFNγ assay (Table 5). The cells were cultured in 200 μL of complete T cell medium. Experimental conditions (duplicates) were as follow: 2×105 total cells per well when cultured in presence of various pAg (10 μM) or medium-only; and 2×104 total cells when cultured in presence of CD3/CD28-loaded bead particles (T cell Activation/Expansion kit, 130-093-627, Miltenyi) (bead-to-cell ratio of 1:1). The cultures were assessed for their capacity to secrete IFNγ (Diaclone Kit Murine IFNγ ELISpot, 862.031-005PC), following the manufacturer's instructions (˜16-18 h incubation time before performing the assay). The peptides used for restimulation are described in Table 5.

TABLE-US-00007 TABLE 5 Setup of the ELISPOT-IFNγ assay. Group Stimulus Wells Animal Total 1 CD22-H1 (10 μM) 3 6 18 CD22-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 2 CD19-H1 (10 μM) 3 6 18 CD19-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 3 CD19-H2 (10 μM) 3 6 18 CD19-B2 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 4 CD37-H1 (10 μM) 3 6 18 CD37-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 5 TNFRSF13C-H1 (10 μM) 3 6 18 TNFRSF13C-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 6 CD22-H1 (10 μM) 3 6 18 CD22-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 7 TNFRSF13C-H1 (10 μM) 3 6 18 TNFRSF13C-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 8 CD37-H1 (10 μM) 3 6 18 CD37-B1 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18 9 MS4A1-B4 (10 μM) 3 6 18 MS4A1-H4 (10 μM) 3 6 18 CD3/CD28 bead 3 6 18 Medium 3 6 18

[0487] Spots were counted on a CTL ELISpot reader. Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).

B. Results

[0488] All mice were aged of 8 to 13 weeks at the experiment starting date. Both males and females were used in the study. Animals have been housed in groups of 6 per cage at maximum. At time of sacrifice, the spleen T cell population was analysed by flow cytometry, showing that the large majority belonged to the CD4+ T cell subset.

[0489] After plating and incubation with the appropriate stimuli, the IFNγ-producing cells were revealed and counted. The data were provided as a number of spots per 1.Math.10.sup.6 total T cells. The individual average values (obtained from the triplicates) were next used to plot the group average values. Statistical analysis for comparison (to the medium condition) were performed using unpaired non-parametric test (Mann Whitney) (**: p<0.01; *: p<0.05).

[0490] Overall, vaccination with the antigenic peptides according to the present invention (CD19-B1, CD19-B2, CD22-B1, CD37-B1, TNFRSF13C-B1 and MS4A1-B4) induced significant T cell responses in the ELISPOT-IFNγ assay in HHD DR1 mice (FIGS. 6-10). Immunogenicity of CD22-B1, CD37-B1, TNFRSF13C-B1 and MS4A1-B4 were confirmed in HHD DR1 mice (FIGS. 11-13).

[0491] The results (FIG. 6) show that immunization of HHD-DR1 mice with CD22-B1 allows to induce T-cells that are able to react strongly after challenge with either CD22-B1 or the human corresponding peptide CD22-H1. Thus, CD22-B2 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0492] These results were confirmed in HHD DR3 mice expressing human HLA-A2 and HLA-DRs MHC and lacking the murine H-2 class I and class II MHCs (FIG. 11).

[0493] The results (FIG. 7) show that immunization of HHD-DR1 mice with TNFRSF13C-B1 allows to induce T-cells that are able to react strongly after challenge with either TNFRSF13C-B1 or the human corresponding peptide TNFRSF13-H1. Thus, TNFRSF13C-B1 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0494] These results were confirmed in HHD DR3 mice expressing human HLA-A2 and HLA-DRs MHC and lacking the murine H-2 class I and class II MHCs (FIG. 12).

[0495] The results (FIG. 8) show that immunization of HHD-DR1 mice with CD37-B1 allows to induce T-cells that are able to react strongly after challenge with either CD37-B1 or the human corresponding peptide CD37-H1. Thus, CD37-B2 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0496] These results were confirmed in HHD DR3 mice expressing human HLA-A2 and HLA-DRs MHC and lacking the murine H-2 class I and class II MHCs (FIG. 13).

[0497] The results (FIG. 9) show that immunization of HHD-DR1 mice with CD19-B2 allows to induce T-cells that are able to react strongly after challenge with either CD19-B2 or the human corresponding peptide CD19-H2. Thus, CD19-B2 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0498] The results (FIG. 10) show that immunization of HHD-DR1 mice with CD19-B1 allows to induce T-cells that are able to react strongly after challenge with either CD19-B1 or the human corresponding peptide CD19-H1. Thus, CD19-B1 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0499] The results (FIG. 15) show that immunization of HHD-DR1 mice with MS4A1-B4 allows to induce T-cells that are able to react strongly after challenge with either MS4A1-B4 or the human corresponding peptide MS4A1-H4. Thus, MS4A1-B4 is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide.

[0500] These results were confirmed in HHD DR3 mice expressing human HLA-A2 and HLA-DRs MHC and lacking the murine H-2 class I and class II MHCs (FIG. 16).

[0501] Altogether, these immunogenicity studies described in Examples 2 performed in HHD DRs and HHD DR1 mice showed that the 6 antigenic peptides of the invention, CD19-B1, CD19-B2, CD22-B1, CD37-B1, TNFRSF13C-B1 and MS4A1-B4 induced strong immune responses. Cross-reactivity of the T cells generated against CD19-B1, CD19-B2, CD22-B1, CD37-B1, TNFRSF13C-B1 and MS4A1-B4 for the corresponding human peptides was shown in HHD DR3 and HHD DR1 mice.

[0502] Accordingly, those results provide experimental evidence that antigen-based immunotherapy is able to improve T cell response in vivo and that the antigenic peptides according to the present invention are particularly efficient for that purpose.

Example 3: Ex Vivo Cytotoxic Effects of CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4 Specific CD8 Human T Cells

[0503] Multiple investigations support the notion of presence of a repertoire of specific T cells against microbial peptides. The number of microbial specific T-cells against peptides is expected to be low, but sufficient to be re-activated by a vaccine challenge.

[0504] To identify and functionally characterize circulating CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4 specific T cells in humans, an in vitro amplification protocol has been developed in order to detect T cells specific for each antigenic peptide and investigate their cytotoxic capacity.

3.1 Identification of Antigenic Peptide-Specific CD8 T Cells in Human

[0505] In vitro amplification method and specific pMHC multimers have been used for identification of CD22-B1, CD37-B1, TNFRSF13C-B1 and MS4A1-B4 specific T cells. pMHC multimers were generated for all the bacteria peptides and their respective human counterpart. PBMCs from several HLA-A*02 healthy donors (up to 19 donors) were collected, enriched after CD137 and CD8 selection and subjected to multiple rounds of in vitro amplification with EO2463 peptides loaded T2 cells to increase the number of specific T cell clones. Detection of OMP peptide specific CD8 T cells using cytometry analysis with the fluorescent multimer was performed on enriched CD8 T cell populations

[0506] FIG. 17 exemplifies results obtained with one HLA-A2 healthy donor. For this donor, cell amplification allows detection of MS4A1-B4 specific cells (19.7%), TNFRSF13C-B1 specific cells (13%), CD22-B1 specific cells (4.6%) and CD37-B1 specific cells (2.5%).

[0507] In conclusion, these results demonstrate the presence of CD8 T cells in the blood of healthy HLA-A2 donors that can recognize the microbiome-derived peptides, and importantly also the human counterpart peptides.

3.2 Antigenic Peptide-Specific CD8 T Cytotoxicity Functions

[0508] CD8+ T cells expanded per above were used to perform cytotoxic assays in presence of different ratios of target and effector cells to assess their cytotoxic capacity, using flow cytometry readout. Target cells were T2 cell lines loaded with bacterial peptide or human counterpart peptide. Negative control was T2 cells unloaded and T2 cells loaded with irrelevant peptide. As shown in FIG. 18, antigenic peptide-specific human T cells clone expanded in vitro have the capacity to kill T2 cells loaded with all the bacteria peptide, CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4. More importantly, CD22-B1, CD37B1, TNFRSF13C-B1 and MS4A1-B4-specific human T cells clones expanded in vitro were able to kill T2 cells loaded with the human TNFR13C (BAFF-R) or MS4A1 (CD20) peptide when cross-reactivity is observed with the staining (FIG. 17).

[0509] Overall, these results demonstrate the presence of T cell clones in healthy volunteers able to recognize microbial peptide and to kill target with microbial peptides and human counterparts. These data are particularly encouraging as T cell clones have been obtained in healthy donors, therefore we could expect that specific T cell clones could be efficiently amplified in patients exposed to the immunization by antigenic peptides of the invention.

Example 4: Further Antigenic Peptides have Superior Affinity to the HLA-A*0201 Allele

[0510] Next, binding affinity of further selected antigenic peptides and of the corresponding fragments of human tumor antigens (human reference peptides) to the HLA-A*0201 allele was confirmed in vitro.

[0511] Namely, the antigenic peptides of sequence SEQ ID NO: 110 («YIFEHPELL» also referred to herein as CD22-B1), of sequence SEQ ID NO: 107 («LIFEHPERV» also referred to herein as CD22-B12), and of sequence SEQ ID NO: 108 («RVFEHPELV» also referred to herein as CD22-B13) were compared to the corresponding reference human peptide derived from CD22 («WVFEHPETL», SEQ ID NO: 270, also referred herein as CD22-H1).

[0512] Moreover, the antigenic peptides of sequence SEQ ID NO: 114 («FLAFVPLQL» also referred herein as CD37-B1), of sequence SEQ ID NO: 119 («ILAFVPLYL» also referred to herein as CD37-B12), of sequence SEQ ID NO: 120 («IMAFVPLAV» also referred to herein as CD37-B13), of sequence SEQ ID NO: 491 («FLAFVPLDV» also referred to herein as CD37-B14) and of sequence SEQ ID NO: 493 («VLAFVPLGV» also referred to herein as CD37-B15) were compared to the corresponding reference human peptide derived from CD37 («GLAFVPLQI», SEQ ID NO: 271, also referred herein as CD37-H1).

[0513] Furthermore, the antigenic peptides of sequence SEQ ID NO: 220 («LMFGAPALV» also referred herein as TNFRSF13C-B1), of sequence SEQ ID NO: 212 («FLFGAPASA» also referred to herein as TNFRSF13C-B11), of sequence SEQ ID NO: 217 («LLFGAPAGV» also referred to herein as TNFRSF13C-B12), and of sequence SEQ ID NO: 224 («VLFGAPAYL» also referred to herein as TNFRSF13C-B13) were compared to the corresponding reference human peptide derived from TNFRSF13C («LLFGAPALL», SEQ ID NO: 279, also referred herein as TNFRSF13C-H1).

[0514] In addition, the antigenic peptides of sequence SEQ ID NO: 65 («AMNSLSLYI» also referred herein as MS4A1-B4), of sequence SEQ ID NO: 70 («YMNSLSLAL» also referred to herein as MS4A1-B42) and of sequence SEQ ID NO: 477 («AMNSLSLTV» also referred to herein as MS4A1-B43) were compared to the corresponding reference human peptide derived from MS4A1 (also known as CD20) («IMNSLSLFA», SEQ ID NO: 264, also referred herein as MS4A1-H4).

A. Materials and Methods

A1. Measuring the Affinity of the Peptide to T2 Cell Line.

[0515] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP ½ negative and incapable of presenting endogenous peptides.

[0516] T2 cells (5.104 cells per well) are incubated with decreasing concentrations of peptides from 100 μM to 0.1 μM (4 points: 100 μM, 10 μM, 1 μM, 0.1 μM) in serum-free medium (TexMacs) supplemented with 100 ng/μl of 32 Microglobulin at 37° C. for 16 hours. Cells are then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).

[0517] The analysis is achieved by FACS (Macsquant analyzer 10-Miltenyi).

[0518] For each peptide concentration, the geometric mean of the labelling associated with the peptide of interest is subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM.

A2. Solubilisation of Peptides

[0519] Each peptide is solubilized by taking into account the amino acid composition. For peptides which do not include any Cystein, Methionin, or Tryptophane, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are resuspended in water or PBS pH7.4.

B. Results

[0520] Results are shown in FIGS. 19-22. For each of the tested human reference epitopes CD22-H1 (FIG. 19), CD37-H1 (FIG. 20), TNFRSF13C-H1 (FIG. 21) and MS4A1-H4 (FIG. 22), the respective antigenic peptides according to the present invention show a stronger binding affinity to HLA-A*0201.

[0521] In summary, the results show that the antigenic peptides according to the present invention show stronger binding affinity to HLA-A*0201 than the corresponding human tumor antigen fragments. As outlined above, without being bound to any theory it is assumed that such a strong binding affinity of the antigenic peptides according to the present invention reflects their ability to raise an immune response (i.e., their immunogenicity).