Immunogenic Polypeptide Composed of HLA-B7 Restricted Tumor Antigen-Derived Optimized Cryptic Peptides, and Uses Thereof

Abstract

The invention pertains to an optimized chimeric polypeptide for use in HLA-337 cancer patients, which comprises four optimized peptides derived from cryptic tumor epitopes (CEA, TERT, MAGE and HER-2/neu) to enhance their immunogenicity.

Claims

1. A polypeptide characterized in that it comprises the sequence SPRLQLSNLXXXAPRRLVQLLXXXGPRALVETLXXXAPKHSDCLA (Seq ID No: 24), wherein the CEA.sub.188L9 (SEQ ID No: 7), TERT.sub.444A1 (SEQ ID No: 9), MAGE.sub.273L9 (SEQ ID No: 10) and HER-2/neu.sub.246A1 (SEQ ID No: 8) epitopes are separated by spacers XXX, in which X is any amino acid or none.

2. The polypeptide according to claim 1, which comprises the sequence SPRLQLSNLAPRRLVQLLGPRALVETLAPKHSDCLA (Seq ID No: 23).

3. The polypeptide according to claim 1 or claim 2, which consists of the sequence SPRLQLSNLAPRRLVQLLGPRALVETLAPKHSDCLA (Seq ID No: 23).

4. The polypeptide according to any of claims 1 to 3, further comprising an endoplasmic reticulum-translocating signal sequence at its N-terminal extremity.

5. The polypeptide according to any of claims 1 to 4, further comprising ubiquitin at its C-terminal extremity.

6. The polypeptide according to any of claims 1 to 5, characterized in that it induces a CD8+ T cells response against at least two epitopes selected from the group consisting of CEA.sub.188, HER-2/neu.sub.246, TERT.sub.444, MAGE.sub.273 A6, MAGE.sub.273 V6 and MAGE.sub.273 I6, in a majority of HHD mice vaccinated with said polypeptide.

7. The polypeptide according to any of claims 1 to 6, characterized in that it induces a CD8+ T cells response against at least two epitopes selected from the group consisting of CEA.sub.188, HER-2/neu.sub.246, TERT.sub.444, MAGE.sub.273 A6, MAGE.sub.273 V6 and MAGE.sub.273 I6 in an in vitro assay with human PBMC from healthy HLA-B*0702 donors.

8. An isolated dendritic cell loaded with a polypeptide according to any of claims 1 to 7.

9. A complex comprising a peptide delivery vector and a polypeptide according to any of claims 1 to 7.

10. A pharmaceutical composition comprising a polypeptide according to any of claims 1 to 7 and/or a dendritic cell according to claim 8 and/or a complex according to claim 9.

11. A polypeptide according to any of claims 1 to 7, and/or a dendritic cell according to claim 8, and/or a complex according to claim 9, for use in cancer immunotherapy in a patient having an HLA-B*0702 phenotype.

12. A kit of parts comprising at least one dose of polypeptide according to any of claims 1 to 7 and at least one dose of adjuvant.

13. A kit of parts comprising at least two doses of polypeptide according to any of claims 1 to 7.

14. The kit of parts according to claim 12 or claim 13, comprising 6 to 20 doses of polypeptide according to any of claims 1 to 7.

15. The kit of parts according to any of claims 12 to 14, wherein each dose of polypeptide comprises between 0.5 and 10 mg of polypeptide.

Description

EXAMPLES

[0042] The examples have been performed using the following materials and methods:

[0043] Transgenic Mice. The HLA-B7 H-2 class-I knockout mice were previously described (Rohrlich et al, 2003) and kindly provided by F. Lemonnier (Institut Pasteur, Paris, France).

[0044] Cells. HLA-B*0702 transfected human T2-B7 cells were previously described (Rohrlich et al, 2003). Cells were grown in penicillin streptomycin FCS 20% supplemented RPMI1640 culture medium.

[0045] Peptides and Plasmids. Peptides were synthesized either by Millegen (Labge, France) or Eurogentec (Seraing, Belgique).

[0046] CTL Induction in vivo in HLA-B*0702 Transgenic Mice. HLA-B*0702 transgenic mice were vaccinated subcutaneously at the base of the tail with 200 g of optimized dipeptides or 400 g of Vbx-016 in association with the HV core T13L helper (150 g) helper peptide and emulsified in Incomplete Freund Adjuvant (IFA) or Montanide ISA51VG (Seppic, Castres, France) twice at two weeks interval.

[0047] Seven days after the last vaccination, spleens were removed and T cells were isolated by Ficoll centrifugation and tested ex vivo for the recognition of 10 M of either the native or optimized peptides. Positive control was Concanavalin A and negative control was medium alone. All conditions were tested in quadriplicates. IFN producing T cells was quantified by ELISpot using the Diaclone Kit Murine IFN ELISpot.

[0048] An immune response was considered positive when there was 1) more than 10 spots difference/10.sup.6 cells between the negative control and the tested peptide and 2) a statistically significant difference between these two groups with T Test (p<0.05).

[0049] Generation of CTL from human PBMC. PBMC were collected by leukapheresis from healthy HLA-B*0702 volunteers. Dendritic cells (DC) were produced from adherent cells cultured for seven days with 500 IU/ml GM-CSF and 500 IU/ml IL-4, in complete synthetic medium (AIMV). On day 6, DCs were pulsed with 10 M of polypeptides overnight. On day 7, CD8+ cells were purified by negative selection with CD8 Dynabeads Untouched Human CD8. CD8+ cells (210.sup.5)+CD8 cells (610.sup.4) were stimulated with 310.sup.4 peptide-pulsed DC in AIMV medium supplemented with 1000 IU/ml IL-6 and 5 IU/ml IL-12, 1 g/ml anti-CD40 and 500 IU/ml IFN in round-bottomed 96-well plates.

[0050] From day 7, cultures were restimulated weekly with peptide-loaded DC in the presence of 20 IU/ml IL-2 and 10 ng/ml IL-7, 1 g/ml anti-CD40 and and 500 IU/ml IFN.

[0051] After the third restimulation, CD8 cells were maintained in AIMV supplemented with 20 IU/ml IL-2 during 7 days. Cultures were starved during one night with AIMV and then were tested in an IFN ELISpot assay.

[0052] IFN ELISpot Assay. For one culture of 96 wells, four pools of 24 wells were collected, CD8 cells were counted and 50 000 CD8 per well were dispatched on the ELISpot plate with 25 000 T2B7 loaded eiher with 10 M of each native monopeptide or with 10 M of modified monopeptides composing the polypeptide. Eight replicates of each condition were performed. Positive control was Phytohaemagglutinin A and negative control was T2B7 loaded with an irrelevant peptide not able to bind HLA-B7 molecules.

Example 1

Evaluation of T Cell Immune Response Generated in HLA-B*0702 Transgenic Mice after Two Vaccinations with all Different Combinations of Dipeptides

[0053] Each dipeptide was used to vaccinate HLA-B*0702 transgenic mice twice at 2 weeks interval and tested for its capacity to induce an immune response against both optimized peptides and their cognate native counterparts. Dipeptides are described in table 2. In each experiment, the best combination is highlighted in grey. 7C1HN3M, 7C1T5M, 7T5HN3M, 7M1HN3M and 7T5M1M gave better results than the inverted sequence, in terms of number of responding mice. 7C1M1M and 7M1C1M gave the same results.

Example 2

Evaluation of T cell Immune Response Generated in HLA-B*0702 Mice after Two Vaccinations with Vbx-016

[0054] To evaluate if the Vbx-016 (SEQ ID No: 23) determined in the dipeptidic experiments is optimum, HLA-B*0702 transgenic mice were vaccinated subcutaneously at the base of the tail with 400 g of Vbx-016 and 150 g of the HBV core T13L helper peptide emulsified in Incomplete Freund Adjuvant (IFA) or Montanide ISA51VG (Seppic, Castres, France) twice at two weeks interval.

[0055] After two vaccinations, all mice respond to at least one native peptide, and 13/14 vaccinated mice responded to two or more cognate native peptides. Importantly, each native peptide was recognized at least once in one vaccinated mouse.

TABLE-US-00004 TABLE 5 Test of the quadripeptide of SEQ ID No: 23 in HLA-B*0702 transgenic mice Number of responding mice/total number of mice Signifiant T-test (<0.05) Helper + adjuvant Vbx016 + Vbx016 + (IFA or helper + IFA helper + Montanide montanide) CEA188 8/9 5/5 13/14 CEA188L9 8/9 5/5 13/14 HER-2Neu246 1/9 0/5 1/14 HER-2Neu246A1 2/9 0/5 2/14 Tert444 8/9 4/5 12/14 Tert444A1 9/9 5/5 14/14 MageA273A6 2/9 0/5 2/14 MageA273I6 1/9 2/5 3/14 MageA273V6 1/9 1/5 2/14 MageA273L9 7/9 5/5 12/14 Vbx-016 9/9 4/4 14/14 Two or more 8/9 5/5 13/14 native epitopes

Example 3

Evaluation of T Cell Immune Response by Inducing Specific Cytotoxic T Lymphocytes from Human Peripheral Blood Mononuclear Cells In Vitro with the Dipeptides Selected in HLA-B*0702 Transgenic Mice

[0056] To evaluate if the junction present in the polypeptide CEA.sub.188L9/TERT.sub.444A1/MAGE.sub.273L9/HER-2/neu.sub.246A1 validated in transgenic mice are processed efficiently in humans, human PBMC were stimulated with each dipeptide (CEA.sub.188L9/TERT.sub.444A1, TERT.sub.444A1/MAGE.sub.273L9 and MAGE.sub.273L9/HER-2/neu.sub.246A1) according to the protocol described in the methods. Recognition of the cognate native peptides was evaluated by measuring specific IFN producing cells from the CD8+ stimulated by the dipeptide, divided in 4 pools for the test. T test was performed when the mean number of spots obtained for the peptide of interest was superior to the mean number of spots obtained with the irrelevant peptide; when t test value was less than 0.05, the result was considered to be significantly positive and is highlighted in grey in the following table.

[0057] In each donor, CTLs were able to recognize both optimized and cognate native peptides (and the four natives for the MAGE-A.sub.273L9 shared peptide), confirming that each junction is well processed by the proteasome.

Example 4

Induction of Specific Cytotoxic T Lymphocytes from Human Peripheral Blood Mononuclear Cells In Vitro with Vbx-016

[0058] Vbx-016 was finally used to stimulate human PBMC from a healthy donor.

[0059] t test was performed when the mean number of spots obtained for the peptide of interest was superior to the mean number of spots obtained with the irrelevant peptide; when t test value was less than 0.05, the result was considered to be significantly positive and is highlighted in grey in the following table. A polyspecific response was induced and several native peptides were recognized by the induced CTLs, confirming that

REFERENCES

[0060] Anderson K, Cresswell P, Gammon M, Hermes J, Williamson A, Zweerink H (1991) Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis. J Exp Med 174: 489-492 [0061] Babon A, Almunia C, Boccaccio C, Beaumelle B, Gelb M H, Menez A, Maillere B, Abastado J P, Salcedo M, Gillet D (2005) Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector. FEBS Lett 579: 1658-1664 [0062] Bungener L, Idema J, ter Veer W, Huckriede A, Daemon T, Wilschut J (2002) Virosomes in vaccine development: induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens. J Liposome Res 12: 155-163 [0063] Butts C, Murray N, Maksymiuk A, Goss G, Marshall E, Soulieres D, Cormier Y, Ellis P, Price A, Sawhney R, Davis M, Mansi J, Smith C, Vergidis D, Ellis P, MacNeil M, Palmer M (2005) Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 23: 6674-6681 [0064] Doling A M, Ballard J D, Shen H, Krishna K M, Ahmed R, Collier R J, Starnbach M N (1999) Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity. Infect Immun 67: 3290-3296 [0065] Fayolle C, Ladant D, Karimova G, Ullmann A, Leclerc C (1999) Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J Immunol 162: 4157-4162 [0066] Gross D A, Graff-Dubois S, Opolon P, Cornet S, Alves P, Bennaceur-Griscelli A, Faure O, Guillaume P, Firat H, Chouaib S, Lemonnier F A, Davoust J, Miconnet I, Vonderheide R H, Kosmatopoulos K (2004) High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy. J Clin Invest 113: 425-433 [0067] Haicheur N, Bismuth E, Bosset S, Adotevi O, Warnier G, Lacabanne V, Regnault A, Desaymard C, Amigorena S, Ricciardi-Castagnoli P, Goud B, Fridman W H, Johannes L, Tartour E (2000) The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J Immunol 165: 3301-3308 [0068] Ishioka G Y, Fikes J, Hermanson G, Livingston B, Crimi C, Qin M, del Guercio M F, Oseroff C, Dahlberg C, Alexander J, Chesnut R W, Sette A (1999) Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes. J Immunol 162; 3915-3925 [0069] Minev B, Hipp J, Firat H, Schmidt J D, Langlade-Demoyen P, Zanetti M (2000) Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci USA 97: 4796-4801 [0070] Ofuji S, Ikeda M, Tsujitani S, Ikeguchi M, Kaibara N, Yuasa I, Mitsuya K, Katoh M, Ito H (1998) Expression of MAGE-1, MAGE-2 and MAGE-3 genes in human gastric carcinomas; lack of evidence for cytotoxic effects in cases with simultaneous expression of MAGE-3 and HLA-A2. Anticancer Res 18: 3639-3644 [0071] Ogata S, Uehara H, Chen A, Itzkowitz S H (1992) Mucin gene expression in colonic tissues and cell lines. Cancer Res 52: 5971-5978 [0072] Oukka M, Manuguerra J C, Livaditis N, Tourdot S, Riche N, Vergnon I, Cordopatis P, Kosmatopoulos K (1996) Protection against lethal viral infection by vaccination with nonimmunodominant peptides. J Immunol 157: 3039-3045 [0073] Peoples G E, Holmes J P, Hueman M T, Mittendorf E A, Amin A, Khoo S, Dehqanzada Z A, Gurney J M, Woll M M, Ryan G B, Storrer C E, Craig D, Ioannides C G, Ponniah S (2008) Combined clinical trial results of a HER2/neu (E75) vaccine for the prevention of recurrence in high-risk breast cancer patients: U.S. Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Clinical cancer research: an official journal of the American Association for Cancer Research 14: 797-803 [0074] Reese D M, Slamon D J (1997) HER-2/neu signal transduction in human breast and ovarian cancer. Stem Cells 15: 1-8 [0075] Rohrlich P S, Cardinaud S, Firat H, Lamari M, Briand P, Escriou N, Lemonnier F A (2003) HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus. Int Immunol 15: 765-772 [0076] Rosenberg S A, Yang J C, Restifo N P (2004) Cancer immunotherapy: moving beyond current vaccines, Nat Med 10: 909-915 [0077] Slamon D J, Clark G M, Wong S G, Levin W J, Ullrich A, McGuire W L (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177-182 [0078] Van den Eynde B J, van der Bruggen P (1997) T cell defined tumor antigens. Curr Opin Immunol 9: 684-693 [0079] Vonderheide R H, Domchek S M, Schultze J L, George D J, Hoar K M, Chen D Y, Stephans K F, Masutomi K, Loda M, Xia Z, Anderson K S, Hahn W C, Nadler L M (2004) Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 10: 828-839 [0080] Vonderheide R H, Hahn W C, Schultze J L, Nadler L M (1999) The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 10: 673-679