ISOLATED MHC-DERIVED HUMAN PEPTIDES AND USES THEREOF FOR STIMULATING AND ACTIVATING THE SUPPRESSIVE FUNCTION OF CD8+CD45RCLOW TREGS

Abstract

Isolated MHC-derived human peptides, particularly an isolated MHC-derived human peptide including a SDVGE-X-R (SEQ ID NO: 13) 7 amino acids motif that is selected from: NQEESVRFDSDVGEFR (Hpep 1-SEQ ID NO:1), NREEYARFDSDVGEFR (Hpep2-SEQ ID NO:2), NREEYVRFDSDVGEYR (Hpep4-SEQ ID NO:4) and any peptide with a length of 16 amino acids including the SDVGE-X-R (SEQ ID NO: 13) motif and having an amino acid sequence with at least 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. Also, the use of these peptides in methods for inducing an immune tolerance, for preventing or reducing transplant rejection or graft versus host disease (GVHD), and in methods for isolating and expanding a population of CD8.sup.+CD45RC.sup.low Tregs or for expanding a population of CD8.sup.+CD45RC.sup.low Tregs and stimulating its immunosuppressive activity.

Claims

1-15. (canceled)

16. An isolated MHC-derived human peptide which comprises a SDVGE-X-R (SEQ ID NO: 13) 7 amino acid motif and which is selected from the group consisting of NQEESVRFDSDVGEFR (Hpep 1-SEQ ID NO:1), NREEYARFDSDVGEFR (Hpep2-SEQ ID NO:2), NREEYVRFDSDVGEYR (Hpep4-SEQ ID NO:4) and any peptide with a length of 16 amino acids comprising the SDVGE-X-R (SEQ ID NO: 13) motif and having an amino acid sequence with at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4.

17. The isolated MHC-derived human peptide according to claim 16, wherein said peptide is NQEESVRFDSDVGEFR (Hpep 1-SEQ ID NO:1) or a peptide with a length of 16 amino acids comprising the SDVGE-X-R (SEQ ID NO: 13) motif and having an amino acid sequence with at least 80% identity with SEQ ID NO: 1.

18. A nucleic acid molecule that encodes the peptide of claim 16.

19. An immunoconjugate comprising an antibody conjugated or fused to the peptide of claim 16.

20. The immunoconjugate according to claim 19, wherein the antibody is directed against a surface antigen of an antigen presenting cell.

21. A nanoparticle comprising at least one peptide of claim 16.

22. A nanoparticle according to claim 21, wherein said nanoparticle is a liposome.

23. A vaccine composition comprising the peptide of claim 16, or an immunoconjugate comprising an antibody conjugated or fused to said peptide, or a nanoparticle comprising said peptide.

24. A MHC class I multimer loaded with the peptide of claim 16.

25. An antibody that specifically binds to the peptide of claim 16 or to a MHC class I multimer loaded with said peptide.

26. A medicament comprising the peptide according to claim 16, or an immunoconjugate comprising an antibody conjugated or fused to said peptide, or a nanoparticle comprising said peptide, or a vaccine composition comprising said peptide.

27. A method for preventing or reducing transplant or graft rejection or graft versus host disease (GVHD) in a patient in need thereof, said method comprising administering to the patient the peptide according to claim 16, or an immunoconjugate comprising an antibody conjugated or fused to said peptide, or a nanoparticle comprising said peptide, or a vaccine composition comprising said peptide.

28. A method for expanding a population of CD8.sup.+CD45RC.sup.low Tregs and stimulating its immunosuppressive activity, comprising a step of culturing a population of CD8.sup.+CD45RC.sup.low Tregs with a culture medium comprising the peptide of claim 16 in presence of a population of antigen presenting cells (APCs), or with a culture medium comprising a MHC class I multimer loaded with said peptide.

29. A population of CD8.sup.+CD45RC.sup.low Tregs obtained by the method of claim 28.

30. A method for preventing or reducing transplant or graft rejection or graft versus host disease (GVHD) in a patient in need thereof, said method comprising administering to the patient the population of CD8.sup.+CD45RC.sup.low Tregs according to claim 29.

31. A method for isolating and expanding a population of CD8.sup.+CD45RC.sup.low Tregs specifically recognizing the peptide according to claim 16, comprising a step of isolating a population of CD8.sup.+CD45RC.sup.low Tregs by MHC/peptide multimer staining with a MHC class I multimer loaded with said peptide, and then a step of expanding the isolated population of CD8.sup.+CD45RC.sup.low Tregs with polyclonal stimulation.

32. A population of CD8.sup.+CD45RC.sup.low Tregs obtained by the method of claim 31.

33. A method for preventing or reducing transplant or graft rejection or graft versus host disease (GVHD) in a patient in need thereof, said method comprising administering to the patient the population of CD8.sup.+CD45RC.sup.low Tregs according to claim 32.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0185] FIG. 1 is a scheme detailing the designed HLA class II peptides (also referred to as MHC class II peptides).

[0186] FIG. 2 is a scheme depicting the protocol of CD8+ Tregs activation in presence of the MHC class II peptides.

[0187] FIG. 3 shows the CD8+ Treg activation in response to human peptides as analyzed by CD25 and CD69 expression. FIG. 3A is a graph showing the expression of the activation markers CD25 (black) and CD69 (white) in CD8+ Tregs incubated with the MHC class II peptides. Bars represent the ratio between the percentage of marker positive cells after peptide stimulation and percentage of marker positive cells in the control condition without peptide +/−SEM. *p<0.05. n=9 to 14 for each peptide. FIGS. 3B and 3C are representative histograms of the expression of CD25 (FIG. 3B) and CD69 (FIG. 3C) in presence of control peptide, MHC class II peptide (Hpep2) or anti-CD/28 stimulation.

[0188] FIG. 4 is a scheme depicting the protocol of CD8.sup.+ Treg expansion by Hpep2 peptide. CD8 Tregs were stimulated at day 0 and day 7 by syngeneic HLA-A2.sup.+ APCs, Hpep2 peptide, IL-2, IL-15 and CpG ODN. Cytokines were added twice a week.

[0189] FIG. 5 is a graph showing the total Treg fold expansion after 14 days culture with Hpep2 or anti-CD3/anti-CD28 mAbs (versus day 0).

[0190] FIG. 6 shows the suppressive activity of CD8.sup.+CD45RC.sup.low Tregs. FIG. 6A is a graph showing the proportion of dividing CD4.sup.+CD25.sup.− T cells co-cultured with CD8.sup.+CD45RC.sup.low Tregs from the same donor. After 14 days of Hpep2 peptide or polyclonal stimulation, expanded Tregs were tested for suppressive activity on syngeneic CFSE-labeled CD4.sup.+CD25.sup.− T cells stimulated with allogeneic APCs pooled from 3 healthy volunteers, as compared to fresh Tregs. n=2 to 5. FIGS. 6B, 6C, 6D and 6E are representative histograms of CFSE staining in the conditions mentioned above: control condition without CD8.sup.+CD45RC.sup.low Tregs (FIG. 6B); in presence of CD8.sup.+CD45RC.sup.low Tregs expanded with the Hpep2 peptide (FIG. 6C); in presence of CD8.sup.+CD45RC.sup.low Tregs expanded with anti-CD3/anti-CD28 mAbs (polyclonal stimulation) (FIG. 6D); in presence of fresh CD8.sup.+CD45RC.sup.low Tregs (FIG. 6E).

[0191] FIG. 7 is a graph showing that Hpep2-expanded CD8.sup.+CD45RC.sup.low Tregs display similar markers expression compared to polyclonally expanded CD8.sup.+CD45RC.sup.low Tregs. CD8.sup.+ Tregs were expanded for 14 days with Hpep2 or anti-CD3/anti-CD28 mAbs and analyzed by flow cytometry for Foxp3, GITR, IL-10, TGF131, IL-34 and IFNγ expression levels. n=6 to 14.

EXAMPLES

[0192] Materials and Methods

[0193] Material

[0194] Human Samples

[0195] Blood was collected at the Etablissement Francais du Sang (Nantes, France). Heparinized blood samples were taken from healthy volunteers after signing an informed consent approved by the ethical committee of relevant institutions (#N° CPDL-PLER-2018 180). The gender of the donors was not available.

[0196] Methods

[0197] Peptides Libraries

[0198] 16-aa peptides were randomly designed on human MHC-II alleles based on their alignment with rat sequence (Genscript, USA). Purity was >90%. Human peptides were dissolved and conserved as described above and diluted at 120 μg/ml in Texmacs medium for use in vitro.

[0199] Cell Purification

[0200] PBMCs were isolated by Ficoll-Paque density-gradient centrifugation at 2000 rpm for 20 min at room temperature without brake. Remaining red blood cells and platelets were removed using 5 min incubation with a hypotonic solution and centrifugation at 1000 rpm for 10 min at 4° C. For pDC and T cell sorting, B cells, monocytes and NK cells were magnetically depleted (Dynabeads, Invitrogen) by using anti-CD19 (clone: HBI19, eBiociences), anti-CD14 (clone: M5E2, eBiociences) and anti-CD16 (Clone: 3G8, purified) mAbs respectively. Enriched PBMCs were stained with anti-CD45RC-FITC (clone: MT2, IQ-Products), anti-CD8α-PE-Cγ7 (clone: RPT8, eBiociences) and anti-Nrp1-PE (clone: u21-1283, BD Biosciences) for sorting of CD8α.sup.+CD45RC.sup.low Tregs and Neurophilin-1.sup.+ pDCs. Enriched PBMCs were stained with anti-CD3-PeCy7 (clone SK7, BD Biosciences), anti-CD4-PerCP-Cy5.5 (clone: RPA-T4, BD Biosciences) and anti-CD25-APC-Cy7 (clone: M-A251, BD Biosciences) mAbs for sorting of CD4.sup.+CD25.sup.− Teff cells. APCs were obtained from PBMCs by either magnetically depleting T cells with an anti-CD3 (OKT3 purified, 5 μg/ml) mAb (for stimulation of Teff in proliferation assays) or by gating out T cells during sorting using anti-CD3-PE (clone: HIT3a, BD Biosciences) (for activation and expansion tests). FACS ARIA II (BD biosciences, Mountain View, Calif.) was used for sorting. Purity was greater than 98%.

[0201] Peptide Stimulation Assay

[0202] CD8.sup.+ CD45RC.sup.low Treg cells and autologous pDCs from the same healthy HLA-A2.sup.+ donor were co-cultured in serum-free Texmacs medium (Miltenyi Biotec) supplemented with IL-2 (25 U/ml, Proleukin, Novartis), CpG ODN 2006 (0.5 μM) and the different synthesized peptides (120 μg/ml) at a ratio 4:1 of Tregs:pDCs for 5 days. CD8.sup.+CD45RC.sup.low Treg activation was analyzed based on expression of CD69 and CD25 markers. As negative control and in order to normalize the results, a negative peptide was used (ALIAPVHAV). Dapi was used as viability marker. As a positive control, CD8.sup.+ CD45RC.sup.low Tregs were stimulated with anti-CD3 (OKT3, 1 μg/ml) and anti-CD28 mAbs (clone: CD28.2; 1 μg/ml). Results were analyzed using the FACS Canto II cytometer (BD Biosciences) and Flowjo software (Tree Star, Inc. USA, version 10).

[0203] Suppression Assays

[0204] CFSE-labeled CD4.sup.+ CD25.sup.− Teffs and CD8.sup.+ CD45RC.sup.low Tregs from the same healthy volunteer (HLA-A2.sup.+) were co-cultured with a pool of allogeneic APCs from 3 different healthy donors (HLA-A2.sup.−) in RPMI1640 medium supplemented with 5% human AB serum. Culture was done at 1:1:1 ratio (where 1=5×10.sup.4 cells/well) for 5 days in triplicate in a V-bottom 96 well plate. Proliferation of CD4.sup.+CD25.sup.− responder T cells was analyzed by flow cytometry (FACS Canto II BD Biosciences TM) by gating on CD3.sup.+CD4.sup.+ living cells (DAPI negative) and using Flowjo software (Tree Star, Inc. USA, version 10).

[0205] Extracellular and Intracellular Stainings

[0206] For the analysis of GITR (clone: DT5D3, Miltenyi Biotec), Foxp3 (clone:259D/C7, BD Biosciences), IL-10 (clone: JES3-9D7 , BD Biosciences), IL-34 (clone: IC5265P, R&D), TGFβ1 (clone: TW4-9E7, BD Biosciences) and IFNγ (clone: B27, BD Biosciences), CD8.sup.+CD45RC.sup.low Treg cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 5 h in presence of Brefeldin A (10 μg/ml) for the last 4 hours in Texmacs medium (Miltenyi Biotec). In order to select viable cells, Fixable Viability Dye eF506 (ThermoFisher Scientific) was used as viability marker. Fc receptors were blocked before staining (BD Biosciences) and cells were permeabilized with Fix/Perm kit (Ebiociences) for intracellular staining Cell phenotype was analyzed by flow cytometry using the LSR II (BD Biosciences, Mountain View, Calif.) and Flowjo software (Tree Star. Inc. USA, version 10).

[0207] Expansion In Vitro of Human CD8.sup.+CD45RC.sup.low Tregs

[0208] 5×10.sup.5 CD8.sup.+CD45RC.sup.low Tregs and 2×10.sup.6 autologous APCs from HLA-A2.sup.+ healthy donors were seeded in a 24 well plate in Texmacs Medium (Miltenyi Biotec), supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), rhlL-2 (1000 U/ml, Proleukin, Novartis), rhIL-15 (10 ng/ml, Miltenyi Biotec), CpG (0.5 μM), and the different peptides (120 μg/ml) derived from HLA-II molecule (GenScript). As negative control, a negative irrelevant peptide (ALIAPVHAV) was used. As a positive control, CD8.sup.+CD45RC.sup.low Tregs were stimulated with plate-coated anti-CD3 (clone: OKT3, 1 μg/ml) mAb and soluble anti-CD28 mAb (clone: CD28.2; 1 μg/ml). At day 7, Tregs were counted and re-expanded. Cytokines were freshly added twice a week. Finally, at day 14, cells were used for experiments.

[0209] Quantification and Statistical Analysis

[0210] For the peptide activation test, a non-parametric Wilcoxon signed-rank test, comparing column median to a hypothetical value of 1.0, was done. Analyses were made with GraphPad Prism 7 software (GraphPad).

[0211] Results

[0212] Four 16aa peptides were designed from four random human MHC class II alleles based on the sequences of the Du51 peptide (NREEYARFDSDVGEYR) and the overlapping 12-mer peptide Bu31-10 (YLRYDSDVGEYR) both bearing the SDVGEYR motif at C-term end (FIG. 1): Hpep1 (NQEESVRFDSDVGEFR-SEQ ID NO: 1), Hpep2 (NREEYARFDSDVGEFR-SEQ ID NO: 2), Hpep3 (NREEYARFDSDVGVYR-SEQ ID NO: 3) and Hpep4 (NREEYVRFDSDVGEYR-SEQ ID NO: 4). We individually tested these peptides differing at positions 2, 5, 6, 14 or 15 in a 5-days culture assay using and syngeneic CD8.sup.+CD45RC.sup.low Tregs and autologous HLA-A2.sup.+ pDCs from the same individuals in presence of interleukin-2 (IL-2) and CpG oligodeoxynucleotides (or CpG ODN) in serum free Texmacs medium (FIG. 2). As shown on FIGS. 3A-C, CD25 and CD69 expression was upregulated on Tregs following incubation with Hpep1, Hpep2 and Hpep4 peptides. These three peptides share the conserved SDVGE-X-R (SEQ ID NO: 13) 7aa motif while Hpep3 has in addition a valine (V) in place of the glutamic acid (E) at position 14 of the peptide, probably impacting TCR recognition.

[0213] From these peptides, shorter peptides were further identified: NREEYARFD (SEQ ID NO: 5), REEYARFDS (SEQ ID NO: 6), EEYARFDSD (SEQ ID NO: 7), EYARFDSDV (SEQ ID NO: 8), YARFDSDVG (SEQ ID NO: 9), ARFDSDVGE (SEQ ID NO: 10), RFDSDVGEF (SEQ ID NO: 11) and FDSDVGEFR (SEQ ID NO: 12).

[0214] To determine whether CD8.sup.+ CD45RC.sup.low Tregs could be expanded using this MHC-II derived peptide, an expansion protocol was set up using sorted CD8.sup.+CD45RC.sup.low/− Tregs and APCs from the same individual with the Hpep2 peptide in presence of IL-2, IL-15 and CpG ODN and compared to a polyclonal stimulation (anti-CD3/28) in similar conditions (FIG. 4). Expansion with APCs and Hpep2 resulted in a 10-fold expansion of the CD8.sup.+CD45RC.sup.low Tregs in 14 days (FIG. 5), although actual expansion of the small Hpep2-specific Treg fraction present at day 0 may be much higher. Importantly, as shown on FIGS. 6A-E, both Hpep2 and anti-CD3/28 expanded CD8.sup.+CD45RC.sup.low Tregs were efficient at suppressing an allogeneic immune response, similarly to fresh CD8.sup.+CD45RC.sup.low Tregs. Interestingly, peptide-stimulated Tregs tend to be more suppressive than polyclonal Tregs, suggesting the potential benefit of expanding antigen-specific Tregs for therapy. As shown on FIG. 7, no significant differences were observed in the level of expression of Foxp3, GITR, IL-10, IFNγ and IL-34 in Hpep2 or anti-CD3/28 expanded CD8.sup.+CD45RC.sup.low Tregs.

[0215] Altogether, this suggest that human HLA class II peptides bearing the consensus SDVGE-X-R (SEQ ID NO: 13) 7aa motif can efficiently activate and expand suppressive human CD8.sup.+ Tregs.

REFERENCES

[0216] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.