PEPTIDES AND COMBINATIONS OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST HEMATOLOGIC NEOPLASMS AND OTHER CANCERS

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T cell receptors, and other binding molecules.

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, and variant sequences thereof which are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 20, and wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) or induces T cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof, wherein said peptide is not a full-length polypeptide.

2. The peptide according to claim 1, wherein said peptide has the ability to bind to an MHC class I or II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 or CD8 T cells.

3. The peptide or variant thereof according to claim 1, wherein the amino acid sequence thereof comprises a continuous stretch of amino acids according to any one of SEQ ID NO: 1 to SEQ ID NO: 20.

4. An antibody or fragment thereof, that specifically recognizes the peptide or variant thereof according to claim 1.

5. The antibody or fragment thereof of claim 4, which is a monoclonal antibody or fragment thereof.

6. A T cell receptor or fragment thereof, that is reactive with an HLA ligand, wherein said ligand is the peptide or variant thereof according to claim 1 when bound to an MHC molecule.

7. A nucleic acid, encoding for any selected from the group consisting of: the peptide or variant thereof according to claim 1 the antibody or fragment thereof according to claim 4, and the T cell receptor or fragment thereof according to claim 6.

8. A recombinant host cell comprising any selected from the group consisting of: the peptide or variant thereof according to claim 1, the antibody or fragment thereof according to claim 4, the T cell receptor or fragment thereof according to claim 6, and the nucleic acid according to claim 7.

9. The recombinant host cell of claim 8, which is selected from the group consisting of: antigen presenting cell, dendritic cell, T cell, and NK cell.

10. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide or variant thereof according to claim 1.

11. An activated T lymphocyte, produced by the method according to claim 10, that selectively recognizes a cell which presents a polypeptide comprising an amino acid sequence given in claim 1.

12. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: the peptide or variant thereof according to claim 1, the antibody or fragment thereof according to claim 4, the T-cell receptor or fragment thereof according to claim 6, the nucleic acid according to claim 7, the recombinant host cell according to claim 8, and the activated T lymphocyte according to claim 11, or a conjugated or labelled active ingredient, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, wherein it comprises at least 5 to 10 different peptides, each peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, and variant sequences thereof which are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 20, and wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) or induces T cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof, wherein said peptide is not a full-length polypeptide.

14. The pharmaceutical composition according to claim 12, which is a vaccine.

15. A kit comprising: (a) a container comprising a pharmaceutical composition containing any selected from the group consisting of: the peptide or the variant thereof according to claim 1, the antibody or fragment thereof according to claim 4, the T cell receptor or fragment thereof according to claim 6, the nucleic acid according to claim 7, the recombinant host cell according to claim 8, and the activated T lymphocyte according to claim 11, in solution or in lyophilized form; (b) a second container containing a diluent or reconstituting solution for the lyophilized formulation, and (c) instructions for (i) use of the solution or (ii) reconstitution or use of the lyophilized formulation.

16. A method for producing a personalized anti-cancer vaccine, said method comprising: a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from an individual patient; b) comparing the peptides as identified in step a) with a warehouse of peptides which have been pre-screened for immunogenicity and/or over-presentation in tumors as compared to normal tissues; c) selecting at least one peptide from the warehouse that matched a TUMAP identified in said patient; and d) formulating the personalized vaccine based on step c), wherein said warehouse comprises a plurality of peptides or variant sequences according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG. 1: Schematic overview of warehouse peptide selection workflow by mass spectrometric HLA ligandome analysis of primary CLL cells, screening for mutation-derived HLA-presented peptides and comparative immunopeptidome analysis with a large benign tissue-derived HLA ligandome database.

[0121] FIG. 2: Schematic overview of immunogenicity analyses of warehouse peptides based on the detection of spontaneous peptide-specific T cell responses via IFN-γ ELISPOT and intracellular cytokine staining or by in vitro priming using T cells of healthy volunteer and CLL patients by mass spectrometric HLA ligandome analysis of primary CLL cells, screening for mutation-derived HLA-presented peptides and comparative immunopeptidome analysis with a large benign tissue-derived HLA ligandome database.

[0122] FIG. 3: INF-γ ELISPOT immunomonitoring of a colon carcinoma patient before subcutaneous personalized peptide vaccination with XS15 in montanide 5 weeks and 16 weeks after vaccination. The functionality of peptide-specific CD4+ and CD8+ T cells for two vaccine cocktail peptides is demonstrated. A total of two vaccinations (week 1 and week 8) were carried out as part of an individual healing trial. The selection of the peptides was based on a mutation panel analysis and an individual HLA ligandome analysis of the patient's tumor tissue.

[0123] FIG. 4: Influence of the BTK inhibitor ibrutinib on (A) peptide-induced T cell response and (B) the HLA ligandome of CLL. (A) Retrospective analysis of preexisting immune responses directed against HLA class I and HLA class II viral T cell epitopes analyzed in IFN-γ ELISPOT assays after 12-d recall stimulation of PBMCs from CLL patients treated in vitro with ibrutinib or mock control. (B) Volcano plots of immunopeptidome modulations in the relative abundances of HLA ligands on primary CLL cells treated in vitro with ibrutinib versus mock treated cells at t24 h and t48 h, respectively. Each dot represents a specific HLA ligand. Log2-fold-changes of their abundance are indicated on the x-axis, the corresponding significance levels on the y-axis. HLA ligands showing significant up- or down-modulation are highlighted in red and blue. CLL warehouse peptides are marked in black.

[0124] FIG. 5: Study design.

[0125] FIG. 6: CLL peptide warehouse.

EXAMPLES

1. Material and Methods

Patient Samples

[0126] For HLA ligandome analysis, peripheral blood monocular cells from CLL patients were collected at the Department of Hematology and Oncology in Tubingen. Cells were isolated by density gradient centrifugation and stored at 80° C. until further use. Informed consent was obtained in accordance with the Declaration of Helsinki protocol. The study was performed according to the guidelines of the local ethics committees (373/2011B02, 454/2016B02). HLA typing was carried out by the Department of Hematology and Oncology, Tubingen, Germany.

Isolation of HLA Ligands

[0127] HLA class I and II molecules were isolated by standard immunoaffinity purification using the pan-HLA class I-specific W6/32 monoclonal antibody, the pan-HLA class II-specific Tü-39 monoclonal antibody, and the HLA-DR-specific L243 monoclonal antibody to extract HLA ligands.

Analysis of HLA Ligands by LC-MS/MS

[0128] Peptides of HLA ligand extracts were separated by nanoflow high-performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) using a 50 μm×25 cm PepMap rapid separation liquid chromatography column and a gradient ranging from 2.4% to 32.0% acetonitrile over the course of 90 min. Eluting peptides were analyzed in an online-coupled LTQ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectron spray ion source using a data dependent acquisition mode employing a top speed CID fragmentation method (normalized collision energy 35%). Mass range for HLA class I peptide analysis was set to 400-650 m/z with charge states 2+ and 3+ selected for fragmentation. For HLA class II peptide analysis mass range was limited to 400-1,000 m/z with charge states 2+ to 5+ selected for fragmentation.

Data Processing and HLA Annotation

[0129] For data processing the SEQUEST HT search engine was used to search the human proteome as comprised in the Swiss-Prot database (20,279 reviewed protein sequences, Sep. 27, 2013) without enzymatic restriction. Precursor mass tolerance was set to 5 ppm, and fragment mass tolerance to 0.02 Da. Oxidized methionine was allowed as a dynamic modification. The false discovery rate was estimated using the Percolator algorithm and limited to 5% for HLA class I and 1% for HLA class II. Peptide lengths were limited to 8-12 amino acids for HLA class I and to 8-25 amino acids for HLA class II. Protein inference was disabled, allowing for multiple protein annotations of peptides. HLA class I annotation was performed using NetMHCpan 4.0 and SYFPEITHI annotating peptides with percentile rank below 2% and ≥60% of the maximal score, respectively.

Amplification of Peptide-Specific T Cells and IFN-γELISPOT Assay

[0130] Peripheral blood mononuclear cells (PBMCs) were pulsed with 1 μg/mL (class I) or 5 μg/mL (class II) per peptide and cultured for 12 days adding 20 U/mL IL-2 on days 3, 5, and 7. Peptide-stimulated PBMCs were analyzed by ELISPOT assay on day 12.

Priming of Naïve CD8.SUP.+ T Cells

[0131] Priming of peptide-specific CD8+ T cells was conducted using artificial antigen-presenting cells. Therefore, MACS-sorted CD8+ T cells were cultured with IL 2 and IL-7. Weekly stimulation with peptide-loaded artificial antigen-presenting cells and IL 12 was performed four times.

Cytokine Staining

[0132] For intracellular cytokine staining, cells were pulsed with 10 μg/mL of individual peptide and incubated with 10 μg/mL Brefeldin A and 10 μg/mL GolgiStop for 12-16 h. Staining was performed using Cytofix/Cytoperm, PerCP anti-human CD8, Pacific Blue anti-human TNF, FITC anti-human CD107a, and PE anti-human IFN-γ monoclonal antibodies. PMA and ionomycin served as positive control. Samples were analyzed on a FACS Canto II cytometer.

Tetramer Staining

[0133] The frequency of peptide-specific CD8+ T cells after priming was determined by PE/Cy7 anti-human CD8 monoclonal antibody and HLA:peptide tetramer-PE staining. Tetramers of the same HLA allotype containing irrelevant control peptides were used as negative control. Samples were analyzed on a FACS Canto II cytometer.

2. Results

In Vitro Experiments

[0134] Personalized peptide vaccine cocktails are based on the individualized HLA ligandome analysis of the CLL cells of each study patients. To allow for a timely and cost-efficient production of personalized vaccine cocktails for a large patient study cohort the inventors developed a so called “warehouse” concept. This premanufactured peptide warehouse includes, based on inventors' preclinical data, the most frequent and CLL-associated antigens for the most common HLA allotypes.

[0135] Vaccine cocktail composition for each individual patient is than performed based on the results of the HLA ligandome analysis of CLL, providing the proof for the natural presentation of warehouse peptides on CLL cells. The feasibility of this warehouse approach was approved by the first results of an ongoing study (Kowalewski et al. (2014), I.c.), showing for more than 90% of the study patient at least on naturally presented warehouse peptide in the immunopeptidome of CLL cells. Therefore, this warehouse approach will be also applied in a study further demonstrating the benefits of the invention in the clinic.

[0136] The inventors redesigned the CLL warehouse based on the results of preclinical data on 52 HLA ligandomes of primary CLL cells and the first data of a previous trial using a novel innovative workflow as depicted in FIG. 1.

[0137] In contrast to analyses in the art where one could detect naturally presented mutation-derived peptides from specific mutations an in-depth search in or mass spectrometric data for mutation-derived peptides for the most common CLL-specific mutations revealed no naturally presented neoepitopes. Therefore, the inventors focused for the design of the peptide warehouse on non-mutated CLL-associated antigens. These were selected based on a direct comparative immunopeptidome analysis using a proprietary large in-house database including more than 350 immunopeptidomes of various benign tissues including B cells, bone marrow, CD34.sup.+ progenitor cells as well as solid organ tissues. Based on the data of an ongoing clinical study the inventors selected A*02, A*24 and B*07 as most common HLA allotypes in the CLL cohort for the novel peptide warehouse. For these HLA class I allotypes as well as for HLA class II the inventors selected the three and five most frequent CLL-exclusive antigens for the peptide warehouse, respectively.

[0138] With this approach the Inventors developed the peptides comprising the amino acid sequences SEQ ID NOs: 1-20, which are depicted in above table 1.

[0139] In a next step the potential of the warehouse peptides according to the invention to induced functional T cell responses was proven either by characterizing of spontaneous T cell responses against our peptides in CLL patients using IFN-γ ELISPOT and intracellular cytokine staining or by in vitro priming using T cells of healthy volunteer and CLL patients.

[0140] A schematic overview of the immunogenicity analyses is depicted in FIG. 2.

[0141] To induce strong and clinically effective T cell responses in the patient, the combination of peptide-based immunotherapy with appropriate adjuvants is a main prerequisite. So far only the TLR ligand imiquimod locally applied on vaccination side was available for use in clinical peptide vaccination studies. Thus, the inventors characterized in recent years the new water-soluble and thus easily GMP-producible TLR2 ligand XS15. Preliminary work has shown that XS15 can induce a strong CD8.sup.+ and Th1CD4.sup.+ T cell response in vivo after subcutaneous injection in healthy donors and individual tumor patients for viral, mutated and unmutated peptides in a water-oil emulsion (montanide ISA 51) (FIG. 3).

[0142] XS15 results in granuloma formation on vaccination site where the vaccinated peptides persist for at least 7 weeks. Furthermore peptide-specific T cells could also be detected on granuloma site, however with a lower frequency than observed in peripheral blood, which confirms that there is no risk of T cell sequestration, dysfunction or deletion on vaccination site using XS15 in montanide. Strikingly, the induced immune responses persist for more than 1.5 years.

[0143] Ibrutinib represents a new treatment standard in first- and second-line therapy for CLL. Therefore, in an embodiment the inventors' multi-peptide vaccine may be applied in patients under ibrutinib treatment. As described, several groups have postulated the positive effect of ibrutinib on anti-cancer immune response. However, ibrutinib is also proposed as potential agent to treat graft versus host disease after allogeneic stem cell transplantation, due to its inhibitory effect on Th2 cells. Thus, the inventors evaluated in own preclinical work the effect of ibrutinib on peptide-induced T cell response, showing no difference in the IFN-γ production of CLL patient-derived T cells to an EBV (Epstein-Barr virus) peptide mix with and without in vitro application of ibrutinib (FIG. 4A).

[0144] Furthermore, the inventors could show that ibrutinib has no relevant effect on the HLA class I and II expression as well as on the composition of the immunopeptidome of CLL cells, warranting the stable presentation of the CLL-associated peptides according to the invention under ibrutinib treatment (FIG. 4B).

Clinical Study

[0145] The clinical study is an open label phase I/II study evaluating a personalized multi-peptide vaccination in combination with the TLR2 ligand XS15 in CLL patients under ibrutinib treatment. The primary objective of this trial is to evaluate the safety and tolerability of the individually composed multi-peptide vaccine in combination with the TLR2 agonist XS15 in montanide ISA51. Toxicity is determined by evaluation of the number of adverse events according to CTCAE V 5.0. The secondary objective of this trial is to evaluate the immunogenicity of the individually composed multi-peptide vaccine in combination with the adjuvant XS15 as determined by induction of T cell immunity. The induction of peptide-specific T-cell responses will be determined by IFN-γ ELISPOT assays.

[0146] These assays are performed after obtaining and processing of the last blood sample of each individual patient one month (end of treatment visit) and seven months (follow up) after the last peptide vaccination. Further secondary objectives of this trial are progression free survival (PFS) and the remission status as well as the evaluation of achievement of MRD-negativity or MRD level reduction at the end of study. Furthermore, for all patients participating in this study PFS, overall survival (OS) and remission status will be assessed two and five years after the end of the study outside the study protocol. In further exploratory endpoints the inventors will correlate the inducibility of immune responses to the multi-peptide vaccine with clinical, biological and mass spectrometric patient characteristics to develop novel biomarkers predicting the immunological and clinical response to peptide-based immunotherapy.

[0147] Physically fit patients (as defined by ECOG score 2) with a confirmed diagnosis of a CLL or small lymphocytic lymphoma (SLL) according to the IWCLL guidelines who have achieved at least a partial remission (PR) (IWCLL guidelines for the diagnosis and treatment of CLL) under ibrutinib treatment but are still MRD positive (CLL cells in peripheral blood or bone marrow ≥10.sup.−4, determined by flow cytometry) are treated within the study.

[0148] Study inclusion (screening visit) is performed before the start of ibrutinib treatment, to allow for an analysis of individual patients' immunopeptidome of CLL cells. CLL patients included in the study receive ibrutinib as a mono therapy, no combination with chemotherapy, antibody therapy or other small molecules is allowed. Patients included in the study can receive ibrutinib as first line treatment or at disease relapse. Previous CLL specific therapies are assessed during the screening visit. Peptide vaccination takes place in CLL patients that achieved at least a partial remission with detectable MRD after at least two and less than 12 months of ibrutinib treatment. Vaccination takes place every four weeks. A total of three vaccinations are carried out. Vaccines are applied subcutaneously in the abdomen after emulsification of the vaccine cocktail (10 peptides+XS15) in montanide ISA51. Ibrutinib treatment will be continued during peptide vaccination and afterwards (FIG. 5).

Warehouse Production and Peptide Cocktail Formulation

[0149] Multi-peptide vaccine cocktails consist, in an embodiment, of 300 μg of each of CLL-associated peptides. In an embodiment the vaccine cocktail consists of three HLA class I and five HLA class II-restricted CLL-associated peptides as well as one HLA class II-restricted adenovirus (ADV) and one HLA-class II-restricted survivin-derived control peptide. The HLA class I-restricted peptides for each patient are selected individually based on the patient-individual HLA allotypes and immunopeptidome analysis of CLL cells from a premanufactured warehouse consisting of, in an embodiment, 9 different peptides restricted to the 3 most common HLA class I allotypes observed in the CLL patient cohort (A*02, A*24, B*07). Peptides were synthesized in the GMP-certified Wirkstoffpeptidlabor at the University of Tubingen. All peptides are synthetic peptides, which are manufactured by well-established solid phase peptide synthesis (SPPS) procedures using Fmoc chemistry.

[0150] Vaccine cocktail formulation (10 patient-individual warehouse peptides and the TLR-2 ligand XS15 (0.5 mg/ml) in H.sub.2O/DMSO) is performed at the GMP-Center of the University Hospital Tubingen.

[0151] The warehouse peptides comprise the most frequently detected CLL-associated antigens identified in the inventors' experimental and clinical studies, as described above. HLA class I peptides to be applied in each CLL patient are selected in a personalized manner guided by the mass spectrometric analysis of the patient-individual HLA ligandome. Therefore, LC/MS/MS-based immunopeptidome analyses are performed from CLL cells, taken at the screening visit before the start of ibrutinib treatment. In addition, in an embodiment, seven HLA class II peptides (five CLL-associated warehouse peptides and two control peptides) are be administered in every patient (FIG. 6).

3. Conclusion

[0152] The inventors were able to develop peptides which do allow the production of a personalized multi-peptide vaccine against cancer, e.g. hematologic neoplasms such as CLL, a cancer entity mainly affecting elderly patients. The approach according to the invention overcomes the shortcomings in the art, for the following reasons: [0153] Peptide cocktail selection from a premanufacture warehouse will allow for a time and cost saving and therefore broadly applicable individualized vaccine production. [0154] The selection of the warehouse peptides is based on immunopeptidome data from a uniquely large cohort of primary CLL samples (n=52) and the largest immunopeptidome database of healthy tissue (n=350). [0155] The patient-individual selection of peptide vaccines is based on a direct immunopeptidome analysis of each study patients' CLL cells. [0156] The invention is accessible to novel adjuvants, such as the TLR1/2 ligand XS15. This will allow for the induction of long-lasting peptide-specific T cell responses, as already proven in preclinical and first clinical evaluations. [0157] Peptide vaccination can be combined with the standard treatment, such as ibrutinib, for which positive effects on T cell response was reported and does not affect the presentation of CLL-associated peptide targets. [0158] Peptide vaccination can be applied after reduction of leukemia burden in the MRD setting enabling an optimal effector T cell to target cell ratio.

[0159] These particularities overcome limitations of presently available peptide-based strategies and bring T cell-based immunotherapy to the next level, and to improve treatment options for patients with cancer, in particular hematologic neoplasms such as CLL.