NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST ESOPHAGEAL CANCER AND OTHER CANCERS

• US 20170029486A1
• Filed: 07/05/2016
• Published: 02/02/2017
• Est. Priority Date: 07/06/2015
• Status: Active Grant

• Current Assignee: Immatics Biotechnologies GmbH (Immatics NV)
• Original Assignee: Immatics Biotechnologies GmbH (Immatics NV)
• Inventors: MAHR, Andrea, WEINSCHENK, Toni, SONG, Colette, SCHOOR, Oliver, FRITSCHE, Jens, SINGH, Harpreet
• Granted Patent: US 10,011,645 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/001152 Transcription factors, e.g. SOX or c-MYC
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4644 Cancer antigens
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/4748 Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• CPC Class Codes: C07K 14/635 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/2833 against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/0638 Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 33/57484 : involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

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. 93, and variant sequences thereof which are at least 88% homologous to SEQ ID No. 1 to SEQ ID No. 93, wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and/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 or variant according to claim 1, wherein said peptide has the ability to bind to a MHC class-I or -II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/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 the group of SEQ ID No. 1 to SEQ ID No. 93.

4. The peptide or variant thereof according to claim 1, wherein said peptide or variant thereof has an overall length of from 8 to 100, optionally from 8 to 30, and optionally from 8 to 16 amino acids, and optionally wherein the peptide consists or consists essentially of an amino acid sequence according to the group of SEQ ID No. 1 to SEQ ID No. 93.

6. The peptide or variant thereof according to claim 1, wherein said peptide is part of a fusion protein, optionally comprising N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii).

7. A nucleic acid, encoding a peptide or variant thereof according to claim 1, optionally linked to a heterologous promoter sequence.

8. An expression vector capable of expressing the nucleic acid according to claim 7.

9. A recombinant host cell comprising the peptide or variant according to claim 1, a nucleic acid encoding said peptide or variant or an expression vector capable of expressing said nucleic acid, wherein said host cell optionally is an antigen presenting cell optionally a dendritic cell.

10. The peptide or variant thereof according to claim 1, a nucleic acid encoding said peptide or variant or an expression vector capable of expressing said nucleic acid, or a host cell comprising said peptide or variant, capable of being used in medicine.

11. A method for producing the peptide or variant thereof according to claim 1, the method comprising culturing a host cell that presents the peptide or variant, or expresses a nucleic acid encoding said peptide or variant or comprises an expression vector capable of expressing said nucleic acid, and isolating the peptide or variant thereof from the host cell or its culture medium.

12. 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 according to claim 1.

13. An activated T cell, optionally an activated T lymphocyte, produced by the method according to claim 12, that selectively recognizes a cell which presents a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93, and variant sequences thereof which are at least 88% homologous to SEQ ID No. 1 to SEQ ID No. 93.

14. A method for killing target cells in a patient which target cells present a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93, and variant sequences thereof which are at least 88% homologous to SEQ ID No. 1 to SEQ ID No. 93, the method comprising administering to the patient an effective number of activated T cells as defined in claim 13.

15. An antibody, optionally a soluble or membrane-bound antibody, that specifically recognizes the peptide or variant thereof according to claim 1, optionally the peptide or variant are bound to an MHC molecule, and wherein said antibody optionally carries a further effector function, optionally an immune stimulating domain or toxin.

16. A peptide or variant according to claim 1, a nucleic acid encoding said peptide or variant or an expression vector capable of expressing said nucleic acid, or a host cell comprising said peptide or variant, an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant or the antibody optionally a soluble or membrane-bound antibody, that specifically recognizes the peptide or variant capable of being used in the treatment of cancer or in the manufacture of a medicament against cancer, or in a diagnostic method for detection of cancerous cells.

17. The peptide or variant according to claim 16, wherein said cancer is selected from the group of lung cancer, kidney cancer, brain cancer, stomach cancer, colon or rectal cancer, liver cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma (MCC), melanoma, ovarian cancer, esophageal cancer, urinary bladder cancer, endometrial cancer, gall bladder cancer, and bile duct cancer, and other tumors that show an overexpression of a protein comprising a peptide sequence of any of SEQ ID No. 1 to SEQ ID No. 93.

18. A kit comprising:

(a) a container comprising a pharmaceutical composition containing the peptide(s) or the variant according to claim 1, a nucleic acid encoding said peptide or variant or an expression vector capable of expressing said nucleic acid, or a host cell comprising said peptide or variant, an activated T lymphocyte that selectively recognizes a cell which presents said peptide or variant or the antibody optionally a soluble or membrane-bound antibody, that specifically recognizes the peptide or variant, in solution or in lyophilized form;

(b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation;

(c) optionally, at least one more peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93, and

(d) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation.

19. The kit according to claim 18, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

20. The kit according to claim 18, wherein said peptide is selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93.

39. An aptamer that specifically recognizes the peptide or variant thereof according to claim 1, optionally the peptide or variant thereof being bound to an MHC molecule.

5. The peptide comprising an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93, and variant sequences thereof which are at least 88% homologous to SEQ ID No. 1 to SEQ ID No. 93, wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and/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, wherein said peptide is modified and/or includes non-peptide bonds.

21. A method for producing a personalized anti-cancer vaccine for the use as a compound-based and/or cellular therapy for an individual patient, said method comprising:

a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient;

b) comparing the peptides as identified in a) with a warehouse of peptides that have been pre-screened for immunogenicity and/or over-presentation in tumors as compared to normal tissues, wherein said warehouse of peptides is one or more selected from the group consisting of SEQ ID 1-93;

c) selecting at least one peptide from the warehouse that matches a TUMAP identified in the patient; and

d) manufacturing or formulating the personalized vaccine or compound-based or cellular therapy based on c).

22. The method according to claim 21, wherein said TUMAPs are identified by:

a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to a tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and

a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC ligands derived from proteins over-expressed or aberrantly expressed by the tumor.

23. The method according to claim 21, wherein the sequences of MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor sample, and sequencing the eluted ligands.

24. The method according to claim 21, wherein the normal tissue corresponding to the tissue type of the tumor sample is obtained from the same patient.

25. The method according to claim 21, wherein the peptides included in the warehouse are identified based on the following steps:

aa. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, such as microarrays or sequencing-based expression profiling, comprising identify genes that over-expressed in a malignant tissue, compared with a normal tissue or tissues;

ab. Selecting peptides encoded by selectively expressed or over-expressed genes as detected in aa, and

ac. Determining an induction of in vivo T-cell responses by the peptides as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said patient;

or

ba. Identifying HLA ligands from said tumor sample using mass spectrometry;

bb. Performing genome-wide messenger ribonucleic acid (mRNA) expression analysis by highly parallel methods, optionally microarrays or sequencing-based expression profiling, comprising identify genes that over-expressed in a malignant tissue, compared with a normal tissue or tissues;

bc. Comparing the identified HLA ligands to said gene expression data;

bd. Selecting peptides encoded by selectively expressed or over-expressed genes as detected in bc;

be. Re-detecting of selected TUMAPs from bd on tumor tissue and lack of or infrequent detection on healthy tissues and confirming the relevance of over-expression at the mRNA level; and

bf. Determining an induction of in vivo T-cell responses by the peptides as selected comprising in vitro immunogenicity assays using human T cells from healthy donors or said patient.

26. The method according to claim 21, wherein the immunogenicity of the peptides included in the warehouse is determined by a method comprising in vitro immunogenicity assays, patient immunomonitoring for individual HLA binding, MHC multimer staining, ELISPOT assays and/or intracellular cytokine staining.

27. The method according to claim 21, wherein said warehouse comprises a plurality of peptides selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 178.

28. The method according to claim 21, further comprising identifying at least one mutation that is unique to the tumor sample relative to normal corresponding tissue from the individual patient, and selecting a peptide that correlates with the mutation for inclusion in the vaccine or for the generation of cellular therapies.

29. The method according to claim 28, wherein said at least one mutation is identified by whole genome sequencing.

30. A T-cell receptor, optionally soluble or membrane-bound, that is reactive with an HLA ligand, wherein said ligand has at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93.

31. The T-cell receptor according to claim 30, wherein said amino acid sequence is at least 88% identical to SEQ ID No. 1 to SEQ ID No. 93.

32. The T-cell receptor according to claim 30, wherein said amino acid sequence consists any of SEQ ID No. 1 to SEQ ID No. 93.

33. The T-cell receptor according to claim 30, wherein said T-cell receptor is provided as a soluble molecule and optionally carries a further effector function optionally an immune stimulating domain or toxin.

34. A nucleic acid, encoding for a TCR according to claim 30, optionally linked to a heterologous promoter sequence.

35. An expression vector expressing the nucleic acid according to claim 34.

36. A recombinant host cell comprising the nucleic acid according to claim 34 or a nucleic acid encoding an antibody optionally a soluble or membrane-bound antibody, that specifically recognizes the peptide or variant or an expression vector comprising said nucleic acid, wherein said host cell optionally is a T cell or NK cell.

37. A method for producing the T cell receptor according to claim 30, said method comprising culturing a host cell, and isolating said T cell receptor from said host cell and/or its culture medium.

38. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of

a) a peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93;

b) a T-cell receptor reactive with a peptide and/or the peptide-MHC complex according to a);

c) a fusion protein comprising a peptide according to a), and the N-terminal amino acids 1 to 80 of the HLA-DR antigen-associated invariant chain (Ii),

d) a nucleic acid encoding for any of a) to c) or an expression vector comprising said nucleic acid,

e) a host cell comprising the expression vector of d,

f) an activated T-lymphocyte, obtained by a method comprising contacting in vitro T cells with a peptide according to a) expressed on the surface of a suitable antigen presenting cell for a period of time sufficient to activate said T cell in an antigen specific manner, as well as a method to transfer these activated T cells into the autologous or other patients;

g) an antibody, or soluble T-cell receptor, reactive to a peptide and/or the peptide MHC complex according to a) and/or a cell presenting a peptide according to a), and potentially modified by fusion with optionally immune-activating domains or toxins,

h) an aptamer recognizing a peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93, and/or a complex of a peptide selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 93 with a MHC molecule,

i) a conjugated or labelled peptide or scaffold according to any of a) to h) and a pharmaceutically acceptable carrier, and optionally, pharmaceutically acceptable excipients and/or stabilizers.