阅读说明：本技术 用于预测疾病特异性氨基酸修饰用于免疫治疗的可用性的方法 (Methods for predicting the availability of disease-specific amino acid modifications for immunotherapy ) 是由 乌尔·沙欣 于 2018-06-01 设计创作，主要内容包括：本发明涉及用于预测包含疾病特异性氨基酸修饰的肽或多肽(特别是肿瘤相关新抗原)是否包含可用于免疫治疗(例如用于疫苗接种)的表位(特别是肿瘤相关新表位)的方法。本发明的方法特别地可用于提供对患者的肿瘤具有特异性的疫苗,并且因此可用于个体化癌症疫苗的情况。(The present invention relates to methods for predicting whether a peptide or polypeptide comprising a disease-specific amino acid modification, in particular a tumor-associated neo-antigen, comprises an epitope, in particular a tumor-associated neo-epitope, useful in immunotherapy, e.g. for vaccination. The methods of the invention are particularly useful for providing vaccines that are specific for a patient's tumor, and thus in the case of individualized cancer vaccines.)

1. A method for assessing the availability of disease-specific amino acid modifications in a peptide or polypeptide expressed in diseased cells for immunotherapy, the method comprising determining whether the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different MHC classes of molecules and/or react with T cells restricted to different MHC classes when presented in the context of MHC molecules.

2. The method of claim 1, wherein the different classes of MHC molecules are MHC class I and MHC class II molecules, and/or the T cells restricted to different MHC classes are CD4+ T cells and CD8+ T cells.

3. The method of claim 1 or 2, wherein the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications when presented in the context of MHC molecules react with T cells restricted to different MHC classes indicating that the disease-specific amino acid modifications are useful for immunotherapy.

4. A method for assessing the availability of disease-specific amino acid modifications in a peptide or polypeptide expressed in diseased cells for immunotherapy, the method comprising determining whether a fragment of the peptide or polypeptide comprising the disease-specific amino acid modifications reacts with T cells having a different T cell receptor when presented with the same MHC molecule.

5. The method of claim 4, wherein the different T cell receptors are of different clonotypes.

6. The method of claim 4 or 5, wherein a fragment of the peptide or polypeptide comprising the disease-specific amino acid modification reacts with T cells having different T cell receptors when presented with the same MHC molecule indicates that the disease-specific amino acid modification is useful for immunotherapy.

7. A method for assessing the availability of disease-specific amino acid modifications in a peptide or polypeptide expressed in diseased cells for immunotherapy, the method comprising determining whether the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different MHC molecules of the same class and/or react with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class.

8. The method of claim 7, wherein the different MHC molecules of the same class are different MHC class I molecules and/or the different T cells restricted to the same MHC class are different CD8+ T cells.

9. The method of claim 7 or 8, wherein presentation of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification in the context of different MHC molecules of the same class and/or reaction of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class indicates that the disease-specific amino acid modification is useful for immunotherapy.

10. A method for assessing the availability of disease-specific amino acid modifications in a peptide or polypeptide expressed in a diseased cell for immunotherapy, the method comprising determining or more of:

(i) determining whether the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification are presented in the context of different classes of MHC molecules and/or react with T cells restricted to different MHC classes when presented in the context of MHC molecules,

(ii) determining whether a fragment of said peptide or polypeptide comprising said disease-specific amino acid modification reacts with a T cell having a different T cell receptor when presented in the context of the same MHC molecule, and/or

(iii) Determining whether the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification are presented in the context of different MHC molecules of the same class and/or react with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class.

11. The method of claim 10, wherein the different MHC classes are MHC class I and MHC class II molecules, and/or the T cells restricted to different MHC classes are CD4+ T cells and CD8+ T cells.

12. The method of claim 10 or 11, wherein the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications when presented in the context of MHC molecules react with T cells restricted to different MHC classes indicating that the disease-specific amino acid modifications are useful for immunotherapy.

13. The method of any of claims 10-12, wherein the different T cell receptors have different clonotypes.

14. The method of of any one of claims 10-13, wherein a reaction of the fragment of the peptide or polypeptide comprising the disease-specific amino acid modification with a T cell having a different T cell receptor when presented with the same MHC molecule indicates that the disease-specific amino acid modification is useful for immunotherapy.

15. The method of of any one of claims 10-14, wherein the different MHC molecules of the same class are different MHC class I molecules and/or the different T cells restricted to the same MHC class are different CD8+ T cells.

16. The method of of any one of claims 10-15, wherein presentation of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification in the context of different MHC molecules of the same class and/or reaction of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class indicates that the disease-specific amino acid modification is useful for immunotherapy.

17. A method for selecting and/or ranking disease-specific amino acid modifications for their availability in immunotherapy, the method comprising the steps of:

(i) identifying peptides and/or polypeptides expressed in diseased cells, each peptide and/or polypeptide comprising at least disease-specific amino acid modifications, and

(ii) determining whether the same or different fragments of the peptide or polypeptide comprising the same disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or react with T cells restricted to different MHC classes when presented in the context of MHC molecules, and

(iii) (iii) repeating step (ii) for at least additional amino acid modifications identified in (i).

18. The method of claim 17, wherein the different MHC classes are MHC class I and MHC class II molecules, and/or the T cells restricted to different MHC classes are CD4+ T cells and CD8+ T cells.

19. The method of claim 17 or 18, wherein the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications when presented in the context of MHC molecules react with T cells restricted to different MHC classes indicating that the disease-specific amino acid modifications are useful for immunotherapy.

20. A method for selecting and/or ranking disease-specific amino acid modifications for their availability in immunotherapy, the method comprising the steps of:

(i) identifying peptides and/or polypeptides expressed in diseased cells, each peptide and/or polypeptide comprising at least disease-specific amino acid modifications, and

(ii) determining whether a fragment of a peptide or polypeptide comprising a disease-specific amino acid modification reacts with a T cell having a different T cell receptor when presented in the context of the same MHC molecule, and

(iii) (iii) repeating step (ii) for at least additional amino acid modifications identified in (i).

21. The method of claim 20, wherein the different T cell receptors have different clonotypes.

22. The method of claim 20 or 21, wherein a fragment of the peptide or polypeptide comprising the disease-specific amino acid modification reacts with T cells having different T cell receptors when presented with the same MHC molecule indicates that the disease-specific amino acid modification is useful for immunotherapy.

23. A method for selecting and/or ranking disease-specific amino acid modifications for their availability in immunotherapy, the method comprising the steps of:

(i) identifying peptides and/or polypeptides expressed in diseased cells, each peptide and/or polypeptide comprising at least disease-specific amino acid modifications, and

(ii) determining whether the same or different fragments of a peptide or polypeptide comprising the same disease-specific amino acid modification are presented in the context of different MHC molecules of the same class and/or react with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class, and

(iii) (iii) repeating step (ii) for at least additional amino acid modifications identified in (i).

24. The method of claim 23, wherein the different MHC molecules of the same class are different MHC class I molecules and/or the different T cells restricted to the same MHC class are different CD8+ T cells.

25. The method of claim 23 or 24, wherein presentation of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification in the context of different MHC molecules of the same class and/or reaction of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class indicates that the disease-specific amino acid modification is useful for immunotherapy.

26. A method for selecting and/or ranking disease-specific amino acid modifications for their availability in immunotherapy, the method comprising the steps of:

(i) identifying peptides and/or polypeptides expressed in diseased cells, each peptide and/or polypeptide comprising at least disease-specific amino acid modifications, and

(ii) determining or more of:

(1) determining whether the same or different fragments of the peptide or polypeptide comprising the same disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or react with T cells restricted to different MHC classes when presented in the context of MHC molecules,

(2) determining whether a fragment of a peptide or polypeptide comprising a disease-specific amino acid modification reacts with a T cell having a different T cell receptor when presented with the same MHC molecule, and/or

(3) Determining whether the same or different fragments of the peptide or polypeptide comprising the same disease-specific amino acid modification are presented in the context of different MHC molecules of the same class and/or react with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class; and

(iii) (iii) repeating step (ii) for at least additional amino acid modifications identified in (i).

27. The method of claim 26, wherein the different MHC classes are MHC class I and MHC class II molecules, and/or the T cells restricted to different MHC classes are CD4+ T cells and CD8+ T cells.

28. The method of claim 26 or 27, wherein the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications are presented in the context of different classes of MHC molecules and/or the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modifications when presented in the context of MHC molecules react with T cells restricted to different MHC classes indicating that the disease-specific amino acid modifications are useful for immunotherapy.

29. The method of any of claims 26-28, wherein the different T cell receptors have different clonotypes.

30. The method of of any one of claims 26-29, wherein a reaction of the fragment of the peptide or polypeptide comprising the disease-specific amino acid modification with T cells having different T cell receptors when presented with the same MHC molecule indicates that the disease-specific amino acid modification is useful for immunotherapy.

31. The method of of any one of claims 26-30, wherein the different MHC molecules of the same class are different MHC class I molecules and/or the different T cells restricted to the same MHC class are different CD8+ T cells.

32. The method of of any one of claims 26-31, wherein presentation of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification in the context of different MHC molecules of the same class and/or reaction of the same or different fragments of the peptide or polypeptide comprising the disease-specific amino acid modification with different T cells restricted to the same MHC class when presented in the context of different MHC molecules of the same class indicates that the disease-specific amino acid modification is useful for immunotherapy.

33. The method of any of claims 17 to 32, wherein the different amino acid modifications tested in step (ii) are present in the same and/or different peptides or polypeptides.

34. The method of any of claims 17 to 33, which includes comparing the scores obtained for different amino acid modifications tested in step (ii).

35. The method of any of claims 1-34, wherein the disease-specific amino acid modification is due to a disease-specific somatic mutation.

36. The method of any of claims 1-35, wherein the disease is cancer and the immunotherapy is an anti-cancer immunotherapy.

37. The method of any of claims 1-36, wherein the immunotherapy comprises administering or more of:

(i) a peptide or polypeptide expressed in diseased cells, the peptide or polypeptide comprising at least disease-specific amino acid modifications,

(ii) (ii) a peptide or polypeptide comprising a fragment of the peptide or polypeptide of (i), said fragment comprising at least disease-specific amino acid modifications, and

(iii) (iii) a nucleic acid encoding the peptide or polypeptide of (i) or (ii).

38. The method of any of claims 1-37, which is useful for providing a vaccine.

39. A method for providing a vaccine comprising the steps of:

(i) identifying or more disease-specific amino acid modifications predicted to be useful in immunotherapy by the method of any one of claims 1-38;

(ii) providing a vaccine comprising or more of:

(1) a peptide or polypeptide expressed in diseased cells comprising at least of the disease-specific amino acid modifications predicted to be useful for immunotherapy,

(2) (ii) a peptide or polypeptide comprising a fragment of said peptide or polypeptide in (i), said fragment comprising at least of said disease-specific amino acid modifications predicted to be useful in immunotherapy, and

(3) (iii) a nucleic acid encoding the peptide or polypeptide of (i) or (ii).

40. The method of of any one of claims 1-39, wherein the fragment is an MHC binding peptide or a potential MHC binding peptide or is processable to provide an MHC binding peptide or a potential MHC binding peptide.

41. A vaccine produced according to the method of of any one of claims 38 to 40.

Technical Field

The present invention relates to methods for predicting whether a peptide or polypeptide comprising a disease specific amino acid modification (particularly a tumour associated neo-antigen) comprises an epitope (particularly a tumour associated neo-epitope) that is useful in immunotherapy (e.g. for vaccination). The methods of the invention are particularly useful for providing vaccines that are specific for a patient's tumor, and thus in the case of individualized cancer vaccines.

Background

The evolution of the immune system produces a highly effective network in vertebrates based on two types of defense (innate and adaptive immunity). In contrast to the evolutionarily ancient innate immune system, which relies on invariant receptors (invariators) recognizing common molecular patterns associated with pathogens, adaptive immunity is based on highly specific antigen receptors and clonal selection (clonal selection) on B cells (B lymphocytes) and T cells (T lymphocytes). B cells elicit a humoral immune response by secreting antibodies, while T cells mediate a cellular immune response that results in the destruction of the recognized cells.

T cells play an important role in cell-mediated immunity in humans and animals. Recognition and binding of specific antigens is mediated by T cell receptors expressed on the surface of T cells. T Cell Receptors (TCRs) of T cells are capable of interacting with immunogenic peptides (epitopes) that bind to Major Histocompatibility Complex (MHC) molecules and are presented on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells. To be able to target multiple antigens, the T cell receptors need to be of great diversity.

More and more pathogen-associated antigens and tumor-associated antigens have been identified leading to a broad set of suitable targets for immunotherapy active or passive immunization strategies can specifically target cells presenting immunogenic peptides (epitopes) derived from these antigens.

Tumor vaccines aim to induce endogenous tumor-specific immune responses by active immunization. Different antigenic formats can be used for tumor vaccination, including intact diseased cells, proteins, peptides or immune vectors (e.g., RNA, DNA or viral vectors), which can be applied directly in vivo or in vitro by pulsing Dendritic Cells (DCs) and then transferring into the patient.

Somatic mutations in cancer are ideal targets for therapeutic vaccine approaches (Castle, J.C. et al cancer Res.72, 1081-.&Schreiber, R.D. Science 348, 69-74 (2015)), T ü reci, O. et al Clin.cancer Res.22, 1885-1896(2016)) which can be processed into peptides, presented on the surface of tumor cells and recognized as new epitopes by T cells newly emerged data, which are excluded from central immune tolerance and absent in healthy tissue, thus combining potentially strong immunogenicity with lower autoimmune potential, suggest, for example, checkpoint blockade (Rizvi, N.A. et al Science 348, 124-128 (2015); Snyder, A. et al N.Engl. J.Med.371, 2189-199 (2014); Van Allen, E.M. et al Science 350, 207 (2015), Le, D.T. et al N.Engl. J.J.372, 9 (2015), E.M. M. 35, 2015 10, Ser. No. 35, Ser. 10, No. 11, No. 10, No. 11, No. 10, No. 11, No. 5, No. 11, No. 10, No. 11, No. 5, No. 11, No. 2, No. 5, No. 2, No. 11, No. 5, No. 11, No. 2, No. 5, No. 11, No⁺T cell recognition. Vaccines consisting of novel epitopes predicted by computer (in silico) from the mutant set of data show strong anti-tumor activity and induce complete rejection of established, invasively growing mouse tumors (Kreiter, S. et al Nature 520, 692 696 (2015)). Similarly, MHC class I neo-epitopes identified by exome (exome) and transcriptome analysis alone or in combination with mass spectrometry in mouse tumor models appear to be suitable vaccine targets and tumor rejection antigens (Yadav,m. et al Nature 515, 572-576 (2014); gubin, M.M. et al Nature 515, 577-581 (2014)). In summary, these studies have stimulated enthusiasm for new epitopic vaccines (Carreno, B.M. et al Science 348, 803-.&Harari，A.Ann.Transl.Med.4，262(2016)；Katsnelson，A.Nat.Med.22，122-124(2016)；Delamarre，L.，Mellman，I.&Yadav，M.Science 348，760-1(2015))。

In human cancer, the vast majority of cancer mutations are unique to individual patients and therefore require individualized treatment strategies. For each patient, the individual cancer mutation profile (personal cancer profile) needs to be determined by deep sequencing to inform the composition of the individualized tailored vaccine prepared as needed.

We have established a clinical development guideline-compliant method comprising performing the next sequencing for comprehensive identification of individual mutations by routine tumor biopsy, computer prediction of potentially related HLA class I and HLA class II neo-epitopes, and designing and preparing a unique multiple neo-epitope RNA vaccine for each patient the eligible patients start with a shared tumor antigen vaccine consisting of NY-ESO-1 and tyrosinase RNA until their individualized RNA vaccine is released, a total of 13 patients have completed treatment, which shows to be viable, safe and well tolerated.

Clinical evaluation of the cumulative relapsing metastatic events in all patients revealed a highly significant reduction after neo-epitope RNA vaccination compared to the patient's previous medical history, resulting in a highly favorable clinical outcome with sustained progression-free survival. patients with multiple metastases who were only treated for a short time with a neo-epitope vaccine due to rapid tumor progression responded almost immediately to subsequent PD-1 blockade and experienced complete responses.objective tumor regression associated with direct neo-epitope vaccine treatment was noted in two patients of these patients had complete responses to progressive metastases and continued for sustained disease control for 26 months. patients experienced objective tumor responses but developed long-term relapse (late relapse) despite the presence of multispecific, fully functional neo-antigen reactive T cells.

Thus, there is a need for models that predict whether epitopes (particularly neo-epitopes) will be useful for immunotherapy.

Subtyping of the response specific to the neoepitope not only confirmed our previous CD4 response to the immunogenic neoepitope⁺The high frequency of T-cell mediated recognition (Kreiter, S. et al Nature 520, 692-696(2015)) but also showed CD8 directed against the quarter neo-epitope for vaccines⁺T cell response. Directed against mutated CD4⁺The superiority of the response can be explained by the high promiscuity of HLA class I molecules in terms of composition and length of the peptide ligands, whereas the highly specific HLA class I molecules bind sets of limited peptides with a narrow length distribution (Amold, p.y. et al j.immunol.169, 739-49 (2002)). approximately two thirds of the observed CTL responses were against CD4⁺T cells respond to different positions of the corresponding neoepitope with recognized mutations. Since 50% of all neoepitopes contained in the vaccine displayed CD4⁺T cell response, therefore the most likely observed association is not CD4⁺And pure co-occurrence of the new epitopic immune response of CD 8. CTL epitopes covalently linked to a helper epitope are known to be more immunogenic (Shirai, M. et al J. Immunol.152, 549-556 (1994)). The HLAI class carrying the mutation and class II neo-epitopes provide mechanistically favourable conditions for CTL priming (priming) since CD4⁺T is thinCell recognition in cross-presentation of CD8⁺Ligands on DC for T cell neo-epitopes and provide homologous T cell help through CD 40L-mediated DC activation (Schoenberger, s.p., Toes, r.e., van der Vooff, e.i., offfrana, R.&Melief, C.J.Nature 393, 480-3 (1998). related remarks we also found that identical mutations can lead to presentation on different HLAI-like restriction elements and be separated by CD8⁺T cell-recognized neo-epitopes (fig. 3b) again, we found that neo-epitope specific T cells with different TCR clonotypes (clonotype) recognized identical epitope/restriction element complexes these findings indicate that unexpectedly -wide pools of mutation-specific T cells could be recruited by neo-epitope vaccination, and each single mutation exploits the diversity of T cell specificities.

In summary, the findings presented herein indicate that the determination of a suitable individualized neo-epitope vaccine (in particular an individualized RNA neo-epitope vaccine) can open a pan neo-antigen specific T cell pool in cancer patients, enabling efficient targeting of its mutant group.

Disclosure of Invention

Examples

The techniques and methods used herein are described herein or described in a manner known per se and as described, for example, in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods, including kits and use of reagents, are performed according to the manufacturer's information unless otherwise indicated.