Patent Application
20180030112
The present invention generally relates to histocompatibility antigens, and more specifically to minor histocompatibility antigens (MiHAs) and use thereof, for example in immunotherapies.
While several treatment modalities have proven effective for cancer immunotherapy, cancer immunotherapists will undoubtedly need more than one weapon in their therapeutic armamentarium. In particular, different approaches are required for tumors with high vs. low mutation loads.1 Solid tumors induced by carcinogens (e.g., melanoma, lung cancer) express numerous mutations that create tumor-specific antigens (TSAs) which can be targeted using two approaches: injection of ex vivo expanded tumor-infiltrating lymphocytes and administration of antibodies against checkpoint molecules.1-3 However, TSAs are exceedingly rare on hematologic cancers (HCs), because of their very low mutation load, and alternative targets must therefore be found for immunotherapy of HCs.1 T cells redirected to CD19 or CD20 antigen targets with engineered chimeric antigen receptors are spectacularly effective for treatment of B-cell malignancies and represent a breakthrough in cancer immunotherapy.4,5 However, whether chimeric antigen receptors might be used for treatment of myeloid malignancies remains a matter of speculation.6
Major histocompatibility complex (MHC) molecules are transmembrane glycoproteins encoded by closely linked polymorphic loci located on chromosome 6 in humans. Their primary role is to bind peptides and present them to T cells. MHC molecules (HLA in humans) present thousands of peptides at the surface of human cells. These MHC-associated peptides (MAPs) are referred to as the immunopeptidome. The immunopeptidome of identical twins (AKA syngeneic individuals) is identical. By contrast, MAPs present on cells from HLA-identical non-syngeneic individuals are classified into two categories: i) monomorphic MAPs which originate from invariant genomic regions and are therefore present in all individuals with a given HLA type, and ii) polymorphic MAPs (AKA MiHAs) which are encoded by polymorphic genomic regions and are therefore present in some individuals but absent in other individuals. MiHAs are essentially genetic polymorphisms viewed from a T-cell perspective. MiHAs are typically encoded by bi-allelic loci and where each allele can be dominant (generates a MAP) or recessive (generates no MAP). Indeed, a non-synonymous single nucleotide polymorphism (ns-SNP) in a MAP-coding genomic sequence will either hinder MAP generation (recessive allele) or generate a variant MAP (dominant allele).
Another strategy that can be used for cancer immunotherapy is adoptive T-cell immunotherapy (ATCI). The term “ATCI” refers to transfusing a patient with T lymphocytes obtained from: the patient (autologous transfusion), a genetically-identical twin donor (syngeneic transfusion), or a non-identical HLA-compatible donor (allogeneic transfusion). To date, ATCI has yielded much higher cancer remission and cure rates than vaccines, and the most widely used form of cancer ATCI is allogeneic hematopoietic cell transplantation (AHCT).
The so-called graft-versus-leukemia (GVL) effect induced by allogeneic hematopoietic cell transplantation (AHCT) is due mainly to T-cell responses against host MiHAs: the GVL is abrogated or significantly reduced if the donor is an identical twin (no MiHA differences with the recipient) or if the graft is depleted of T lymphocytes. More than 400,000 individuals treated for hematological cancers owe their life to the MiHA-dependent GVL effect which represents the most striking evidence of the ability of the human immune system to eradicate neoplasia. Though the allogeneic GVT effect is being used essentially to treat patients with hematologic malignancies, preliminary evidence suggests that it may be also effective for the treatment of solid tumors. The considerable potential of MiHA-targeted cancer immunotherapy has not been properly exploited in medicine. In current medical practice, MiHA-based immunotherapy is limited to “conventional” AHCT, that is, injection of hematopoietic cells from an allogeneic HLA-matched donor. Such unselective injection of allogeneic lymphocytes is a very rudimentary form of MiHA-targeted therapy. First, it lacks specificity and is therefore highly toxic: unselected allogeneic T cells react against a multitude of host MiHAs and thereby induce graft-versus-host-disease (GVHD) in 60% of recipients. GVHD is always incapacitating and frequently lethal. Second, conventional AHCT induces only an attenuated form of GVT reaction because donor T cells are not being primed (pre-activated) against specific MiHAs expressed on cancer cells prior to injection into the patient. While primed T cells are resistant to tolerance induction, naïve T cells can be tolerized by tumor cells.
It has been demonstrated in mice models of AHCT that, by replacing unselected donor lymphocytes with CD8+ T cells primed against a single MiHA, it was possible to cure leukemia and melanoma without causing GVHD or any other untoward effect. Success depends on two key elements: selection of an immunogenic MiHA expressed on neoplastic cells, and priming of donor CD8+ T cells against the target MiHA prior to AHCT. A recent report discusses why MiHA-targeted ATCI is so effective and how translation of this approach in the clinic could have a tremendous impact on cancer immunotherapy8.
High-avidity T cell responses capable of eradicating tumors can be generated in an allogeneic setting. In hematological malignancies, allogeneic HLA-matched hematopoietic stem cell transplantation (ASCT) provides a platform for allogeneic immunotherapy due to the induction of T cell-mediated graft-versus-tumor (GVT) immune responses. Immunotherapy in an allogeneic setting enables induction of effective T cell responses due to the fact that T cells of donor origin are not selected for low reactivity against self-antigens of the recipient. Therefore, high-affinity T cells against tumor- or recipient-specific antigens can be found in the T cell inoculum administered to the patient during or after ASCT. The main targets of the tumor-reactive T cell responses are polymorphic proteins for which donor and recipient are disparate, namely MiHAs.
However, implementation of MiHA-targeted immunotherapy in humans has been limited mainly by the paucity of molecularly defined human MiHAs. Based on the MiHAs currently known, only 33% of patients with leukemia would be eligible for MiHA-based ATCI. MiHA discovery is a difficult task because it cannot be achieved using standard genomic and proteomic methods. Indeed, i) less than 1% of SNPs generate a MiHA and ii) current mass spectrometry methods cannot detect MiHAs.
Thus, there is a need for the identification of novel MiHAs that may be used in immunotherapies.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present invention relates to the following items 1 to 53:
1. A Minor Histocompatibility Antigen (MiHA) peptide of 8 to 14 amino acids of the formula I
Z1—X1—Z2 (I)
wherein
Z1 is an amino terminal modifying group or is absent;
X1 is a sequence comprising at least 8 contiguous residues of one of the peptide sequences set forth in Table VI and comprising the polymorphic amino acid depicted; and
Z2 is a carboxy terminal modifying group or is absent;
T/ILLEDGTFKV
R/QLAPTLSQL
I/TLLEDGTFKV
I/SLAPCKLETV
S/ILAPCKLETV
A/SEIEQKIKEY
S/AEIEQKIKEY
R/LLQEELEKL
E/QETAIYKGDY
Q/EEAPESATVIF
N/DEVLIHSSQY
Q/REYQVKLQA
R/QEYQVKLQA
L/M/VEADLPRSW
2. The MiHA peptide of item 1, wherein X1 consists of any one of the peptide sequences set forth in Table VI.
3. The MiHA peptide of item 1, wherein X1 is a sequence comprising at least 8 contiguous residues of one of the peptide sequences set forth in SEQ ID Nos: 1-75 and comprising the polymorphic amino acid.
4. The MiHA peptide of item 3, wherein X1 consists of any one of the peptide sequences set forth in SEQ ID Nos: 1-75.
5. The MiHA peptide of any one of items 1 to 4, wherein Z1 is absent.
6. The MiHA peptide of any one of items 1 to 5, wherein Z2 is absent.
7. The MiHA peptide of any one of items 1 to 6, wherein said MiHA peptide consists of any one of the peptide sequences set forth in Table VI.
8. The MiHA peptide of any one of items 1 to 7, wherein said MiHA peptide consists of any one of the peptide sequences set forth in SEQ ID Nos: 1-75.
9. The MiHA peptide of item any one of items 1 to 8, wherein said MiHA derives from a locus with a minor allele frequency (MAF) of at least 0.1.
10. The MiHA peptide of item 9, wherein said MiHA derives from a locus with a minor allele frequency (MAF) of at least 0.2.
11. The MiHA peptide of any one of items 1 to 10, wherein said MiHA peptide binds to a major histocompatibility complex (MHC) class I molecule of the HLA-A*02:01 allele, and said peptide sequences are set forth in Table VII:
T/ILLEDGTFKV
I/TLLEDGTFKV
I/SLAPCKLETV
S/ILAPCKLETV
R/LLQEELEKL
12. The MiHA peptide of any one of items 1 to 10, wherein said peptide binds to a major histocompatibility complex (MHC) class I molecule of the HLA-B*44:03 allele, and said peptide sequences are set forth in Table VIII:
R/QLAPTLSQL
A/SEIEQKIKEY
S/AEIEQKIKEY
E/QETAIYKGDY
Q/EEAPESATVIF
N/DEVLIHSSQY
Q/REYQVKLQA
R/QEYQVKLQA
L/M/VEADLPRSW
13. A polypeptide comprising an amino acid sequence of at least one of the MiHA peptide defined in any one of items 1 to 12, wherein said polypeptide is of the following formula Ia:
Z1—X2—X1—X3—Z2 (Ia)
wherein
Z1, X1 and Z2 are as defined in any one of items 1 to 12; and
X2 and X3 are each independently absent or a sequence of one or more amino acids, wherein said polypeptide does not comprise or consist of an amino acid sequence of a native protein, and wherein processing of said polypeptide by a cell results in the loading of the MiHA peptide in the peptide-binding groove of MHC class I molecules expressed by said cell
14. A peptide combination comprising (i) at least two of the MiHA peptides defined in any one of items 1 to 12; (ii) at least one of the MiHA peptides defined in any one of items 1 to 12 and at least one additional MiHA peptide.
15. A nucleic acid encoding the MiHA peptide of any one of items 1 to 12 or the polypeptide of item 13.
16. The nucleic acid of item 15, which is present in a plasmid or a vector.
17. An isolated major histocompatibility complex (MHC) class I molecule comprising the MiHA peptide of any one of items 1 to 12 in its peptide binding groove.
18. The isolated MHC class I molecule of item 17, which is in the form of a multimer.
19. The isolated MHC class I molecule of item 18, wherein said multimer is a tetramer.
20. An isolated cell comprising the MiHA peptide of any one of items 1 to 12, the peptide combination of item 14, or the nucleic acid of item 15 or 16.
21. An isolated cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the MiHA peptide of any one of items 1 to 12, or the peptide combination of item 14, in their peptide binding groove.
22. The cell of item 21, which is an antigen-presenting cell (APC).
23. The cell of item 22, wherein said APC is a dendritic cell.
24. A T-cell receptor (TCR) that specifically recognizes the isolated MHC class I molecule of any one of items 17-19 and/or MHC class I molecules expressed at the surface of the cell of any one of items 21-23.
25. One or more nucleic acids encoding the alpha and beta chains of the TCR of item 24.
26. The one or more nucleic acids of item 25, which are present in a plasmid or a vector.
27. An isolated CD8+ T lymphocyte expressing at its cell surface the TCR of item 24.
28. The CD8+ T lymphocyte of item 27, which is transfected or transduced with the one or more nucleic acids of item 25 or 26.
29. A cell population comprising at least 0.5% of CD8+ T lymphocytes according to item 27 or 28.
30. A composition comprising (i) the MiHA peptide of any one of items 1 to 12; (ii) the polypeptide of item 13; (iii) the peptide combination of item 14; (iv) the nucleic acid of item 15 or 16; (iv) the MHC class I molecule of any one of items 17-19; (v) the cell of any one of 20-23; (v) the TCR of item 24; (vi) the one or more nucleic acids of item 25 or 26; the CD8+ T lymphocyte of item 27 or 28; and/or (vii) the cell population of item 29.
31. The composition of item 30, further comprising a buffer, an excipient, a carrier, a diluent and/or a medium.
32. The composition of item 30 or 31, wherein said composition is a vaccine and further comprises an adjuvant.
33. The composition of any one of items 30 to 32, wherein said composition comprises the peptide combination of item 14, or one or more nucleic acids encoding the at least two MiHA peptides present in said peptide combination.
34. The composition of any one of items 30 to 33, which comprises the cell of any one of items 19-22 and the CD8+ T lymphocyte of item 26 or 27.
35. A method of expanding CD8+ T lymphocytes specifically recognizing one or more of the MiHA peptides defined in any one of items 1 to 12, said method comprising culturing, under conditions suitable for CD8+ T lymphocyte expansion, CD8+ T lymphocytes from a candidate donor that does not express said one or more MiHA peptides in the presence of cells according to any one of items 20-22.
36. A method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of (i) the CD8+ T lymphocytes of item 27 or 28; (ii) the cell population of item 29; and/or (iii) a composition comprising (i) or (ii).
37. The method of item 36, said method further comprising determining one or more MiHA variants expressed by said subject in need thereof, wherein the CD8+ T lymphocytes specifically recognize said one or more MiHA variants presented by MHC class I molecules.
38. The method of item 37, wherein said determining comprises sequencing a nucleic acid encoding said MiHA.
39. The method of any one of items 36 to 38, wherein said CD8+ T lymphocytes are ex vivo expanded CD8+T lymphocytes prepared according to the method of item 35.
40. The method of any one of items 36 to 39, wherein said method further comprises expanding CD8+ T lymphocytes according to the method of item 35.
41. The method of any one of items 36 to 40, wherein said subject in need thereof is an allogeneic stem cell transplantation (ASCT) recipient.
42. The method of any one of items 36 to 41, further comprising administering an effective amount of the MiHA peptide recognized by said CD8+ T lymphocytes, and/or (ii) a cell expressing at its surface MHC class I molecules comprising the MiHA peptide defined in (i) in their peptide binding groove.
43. The method of any one of items 36 to 42, wherein said cancer is a hematologic cancer.
44. The method of item 43, wherein said hematologic cancer is leukemia.
45. An antigen presenting cell or an artificial construct mimicking an antigen-presenting cell that presents the MiHA peptide of any one of items 1 to 12 or the peptide combination of item 14.
46. An in vitro method for producing cytotoxic T lymphocytes (CTLs) comprising contacting a T lymphocyte with human class I MHC molecules loaded with the MiHA peptide of any one of items 1 to 12 or the peptide combination of item 14 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 lymphocyte in an antigen-specific manner.
47. An activated cytotoxic T lymphocyte obtained by method of item 46.
48. A method of treating a subject with haematological cancer comprising administering to the patient an effective amount of the cytotoxic T lymphocyte of item 47.
49. A method of generating immune response against tumor cells expressing human class I MHC molecules loaded with the MiHA peptide of any one of items 1 to 12 or the peptide combination of item 14 in a subject, said method comprising administering the cytotoxic T lymphocyte of item 47.
50. An antigen presenting cell (APC) artificially loaded with one or more of the MiHA peptides defined in any one of items 1 to 12, or the peptide combination of claim 14.
51. The APC of item 49 for use as a therapeutic vaccine.
52. A method for generating an immune response in a subject comprising administering to the subject allogenic T lymphocytes and a composition comprising one or more of the MiHA peptides defined in any one of items 1 to 12, or the peptide combination of claim 14.
53. The method of any one of items 48, 49 and 52 wherein said subject has a haematological cancer selected from leukemia, lymphoma and myeloma.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawing:
Terms and symbols of genetics, molecular biology, biochemistry and nucleic acid used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W University Science Books, 2005); Lehninger, Biochemistry, sixth Edition (W H Freeman & Co (Sd), New York, 2012); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. All terms are to be understood with their typical meanings established in the relevant art.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
The terms “subject” and “patient” are used interchangeably herein, and refer to an animal, preferably a mammal, most preferably a human, who is in the need of treatment for cancer using one or more MiHAs as described herein. These term encompass both adults and child.
In an aspect, the present invention provides a polypeptide (e.g., an isolated or synthetic polypeptide) comprising an amino acid sequence of a MiHA peptide, wherein said polypeptide is of the following formula Ia:
Z1—X2—X1—X3—Z2 (Ia)
wherein
Z1, X1 and Z2 are as defined below; and
X2 and X3 are each independently absent or a sequence of one or more amino acids, wherein said polypeptide does not comprise or consist of an amino acid sequence of a native protein (e.g., the amino acid sequence of the native protein from which the MiHA peptide is derived), and wherein processing of said polypeptide by a cell (e.g., an antigen-presenting cell) results in the loading of the MiHA peptide of sequence X1 in the peptide-binding groove of MHC class I molecules expressed by said cell.
In an embodiment, X2 and/or X3 are each independently a sequence of about 1 to about 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500 or 1000 amino acids. In an embodiment, X2 is a sequence of amino acids that is immediately amino-terminal to the sequence of X1 in the native polypeptide from which the MiHA is derived (see Table II for the Ensembl gene ID corresponding to the gene from which the MiHA described herein are derived). In an embodiment, X3 is a sequence of amino acids that is immediately carboxy-terminal to the sequence of X1 in the native polypeptide from which the MiHA is derived (see Table II). For example, MiHA No. 1 derives from the protein Ankyrin repeat domain 13A (ANKRD13A), and thus X2 and/or X3 may comprises the one or more amino acids immediately amino- and/or carboxy-terminal to the sequence SLLESSRSQEL/P (SEQ ID NO: 79) in ANKRD13A (Ensembl gene ID No. ENSG00000076513, NCBI Reference Sequence: NP_149112.1). Thus, the sequences immediately amino- and/or carboxy-terminal to the sequences of the MiHAs described herein may be easily identified using the information available in public databases such as Ensembl, NCBI, UniProt, which may be retrieved for example using the SNP ID Nos. and/or Ensembl gene ID Nos. provided in Table II below. The entire content and information, including the full sequences of the transcripts and encoded polypeptides, corresponding to the SNP ID Nos. and Ensembl gene ID Nos. provided in Table II, are incorporated herein by reference.
In another embodiment, X2 and/or X3 are absent. In a further embodiment, X2 and X3 are both absent.
Thus, in another aspect, the present invention provides a MiHA peptide (e.g., an isolated or synthetic peptide) of about 8 to about 14 amino acids of formula I
Z1—X1—Z2 (I)
wherein Z1 is an amino terminal modifying group or is absent; X1 is a sequence comprising at least 8 (preferably contiguous) residues of one of the peptide sequences of MiHA Nos. 1-93 set forth in Table I below and comprising the polymorphic amino acid (variation) depicted (underlined, e.g., for MiHA No. 1, the C-terminal residue L or P is comprised in X1 and for MiHA No. 2, the C-terminal residue V or L is comprised in domain X1, etc.); and Z2 is a carboxy terminal modifying group or is absent. The reference to MiHA Nos. 1-93 encompasses each of the variants defined by the sequences depicted. For example, the term “MiHA No. 1” (SLLESSRSQEL/P, SEQ ID NO: 79) refers to SLLESSRSQEL (SEQ ID NO: 80) and/or SLLESSRSQEP (SEQ ID NO: 81).
P
T/ILLEDGTFKV
R/QLAPTLSQL
I/TLLEDGTFKV
AGPVHAL
EAQLKRM
RQHW
VKKY
VL
HNLQL
HNLQL
I/SLAPCKLETV
EALDALGLKRY
S/ILAPCKLETV
ITFL
A/SEIEQKIKEY
S/AEIEQKIKEY
R/LLQEELEKL
E/QETAIYKGDY
Q/
EEAPESATVIF
N/DEVLIHSSQY
Q/REYQVKLQA
ERSW
R/QEYQVKLQA
KRSW
L/M/
KRSW
VEADLPRSW
ERSW
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of one of the peptide sequences of MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 (SEQ ID Nos: 1-75), wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 1 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 2 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 3 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence of at least 8 amino acids of MiHA No. 4 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 5 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 6 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 7 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted. In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 8 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 9 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 10 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 11 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 12 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 13 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 14 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 15 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 16 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as define (No. 119 or 120) set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 18 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 19 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 20 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 21 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 22 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 23 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 24 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 25 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 26 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 27 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 28 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 29 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 30 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 31 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 32 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 33 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 34 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 35 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 36 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 37 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 38 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 39 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 40 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 41 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 42 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 43 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 44 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 45 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 46 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 47 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 48 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 49 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 50 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 51 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 52 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 53 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 54 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 55 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 56 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 57 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 58 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 59 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 60 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 61 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 62 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 63 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 64 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 65 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 66 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 67 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 68 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 69 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 70 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 71 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 72 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 73 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 74 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 75 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 76 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 77 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 78 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 79 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 80 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 81 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 82 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 83 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 84 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 85 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 86 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 87 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 88 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 89 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 90 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 91 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 92 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In another aspect, the present invention provides a MiHA peptide of the formula I or Ia as defined above, wherein X1 is a sequence comprising at least 8 amino acids of MiHA No. 93 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted.
In an embodiment, the MiHA peptide is able to bind to, or to be presented by, HLA-A2 molecules (HLA-A*02:01 allele). In another aspect, the present invention provides an HLA-A2-binding MiHA peptide of 8-14 amino acids of the formula I as defined above, wherein X1 is a sequence of at least 8 amino acids of any one of the MiHA Nos. 1, 2, 12, 17-19, 24-28, 30, 32, 49-53, 55, 57, 59-61, 65, 72-75, 85 and 92 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted. In an embodiment, the HLA-A2-binding MiHA peptide comprises or consists of the sequence of MiHA Nos. 32 and 49-50.
In an embodiment, the MiHA peptide is able to bind to, or to be presented by, HLA-B44 molecules (HLA-B*44:03 allele). In another aspect, the present invention provides an HLA-B44-binding MiHA peptide of 8-14 amino acids of the formula I as defined above, wherein X1 is a sequence of at least 8 amino acids of any one of the MiHA Nos. 3-11, 13-16, 20-23, 29, 31, 33-48, 54, 56, 58, 62-64, 66-71, 76-84, 86-91 and 93 set forth in Table I, wherein said sequence comprises the polymorphic amino acid depicted. In an embodiment, the HLA-B44-binding MiHA peptide comprises or consists of the sequence of MiHA Nos. 7, 8, 10, 14-15, 20-21, 33, 35, 39-47, 66, 70-71, 76, 78, 81, 84 and 90.
In an embodiment, the MiHA peptide is derived from a gene that does not exhibit ubiquitous expression. The expression “does not exhibit ubiquitous expression” is used herein to refer to a gene which, according to the data from Fagerberg et al., Mol Cell Proteomics 2014 13: 397-406, is not expressed with a FPKM>10 in all 27 tissues disclosed therein.
In an embodiment, the MiHA peptide derives from a locus with a minor allele frequency (MAF) of at least 0.05 as determined according to data from the dbSNP database (NCBI) and the National Heart, Lung and Blood Institute (NHLBI) Exome Sequencing Project (ESP) (as set forth in Table II). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.1 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.1 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.15 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.15 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.2 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.2 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.25 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.25 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.3 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.3 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.35 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.35 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.4 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). In an embodiment, the MiHA peptide derives from a locus with a MAF of at least 0.4 as determined according to data from the dbSNP database (NCBI) and the NHLBI Exome Sequencing Project (ESP).
In some embodiments, the present invention provides a MiHA peptide comprising any combination/subcombination of the features or properties defined herein, for example, a MiHA peptide of the formula I as defined above, wherein the peptide (i) binds to HLA-A2 molecules, (ii) derives from a gene that does not exhibit ubiquitous expression and (iii) derives from a locus with a MAF of at least 0.1 as determined according to data from the dbSNP database (NCBI) and/or the NHLBI Exome Sequencing Project (ESP).
In general, peptides presented in the context of HLA class I vary in length from about 7 to about 15, or preferably 8 to 14 amino acid residues. In some embodiments of the methods of the invention, longer peptide comprising the MiHA peptide sequences defined herein are artificially loaded into cells such as antigen presenting cells (APCs), processed by the cells and the MiHA peptide is presented by MHC class I molecules at the surface of the APC. In this method, peptides/polypeptides longer than 15 amino acid residues (i.e. a MiHA precursor peptide, such as those defined by formula Ia above) can be loaded into APCs, are processed by proteases in the APC cytosol providing the corresponding MiHA peptide as defined herein for presentation. In some embodiments, the precursor peptide/polypeptide (e.g., polypeptide of formula Ia defined above) that is used to generate the MiHA peptide defined herein is for example 100, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids or less. Thus, all the methods and processes using the MiHA peptides described herein includes the use of longer peptides or polypeptides (including the native protein), i.e. MiHA precursor peptides/polypeptides, to induce the presentation of the “final” 8-14 MiHA peptide following processing by the cell (APCs).
In some embodiments, the above-mentioned MiHA peptide is about 8 to 12 amino acids long (e.g., 8, 9, 10, 11 or 12 amino acids long), small enough for a direct fit in an HLA class I molecule (HLA-A2 or HLA-B44 molecule), but it may also be larger, between 12 to about 20, 25, 30, 35, 40, 45 or 50 amino acids, and a MiHA peptide corresponding to the domain defined by X1 above be presented by HLA molecules only after cellular uptake and intracellular processing by the proteasome and/or other proteases and transport before presentation in the groove of an HLA class I molecule (HLA-A2 or HLA-B44 molecule), as explained above.
In an embodiment, the MiHA peptide consists of an amino acid sequence of 8 to 14 amino acids, e.g., 8, 9, 10, 11, 12, 13, or 14 amino acids, wherein the sequence is the sequences of any one of MiHA Nos. 1-93 set forth in Table I. In another aspect, the present invention provides a MiHA peptide consisting of an amino acid sequence of 8 to 14 amino acids, e.g., 8, 9, 10, 11, 12, 13, 14 or 15 amino acids, said amino acid sequence consisting of the sequence of MIHA Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 of Table I). In an embodiment, the at least 7 or 8 amino acids of one of MIHA Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 of Table I are contiguous amino acids. In an embodiment, X1 is a domain comprising at least 8 amino acids of any one of MiHA Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90, wherein said sequence comprises the polymorphic amino acid depicted. In another embodiment, X1 is a sequence comprising, or consisting of, the amino acids of any one of MiHA Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90.
The term “amino acid” as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of MiHA peptides. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc.
Other amino acids include for example non-genetically encoded forms of amino acids, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include, for example, beta-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry. In an embodiment, the MiHA peptide comprises only naturally-occurring amino acids.
In embodiments, the MiHA peptides of the present invention include peptides with altered sequences containing substitutions of functionally equivalent amino acid residues, relative to the above-mentioned sequences. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (having similar physico-chemical properties) which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions.
The above-mentioned MiHA peptide may comprise all L-amino acids, all D-amino acids or a mixture of L- and D-amino acids. In an embodiment, the above-mentioned MiHA peptide comprises all L-amino acids.
The MiHA peptide may also be N- and/or C-terminally capped or modified to prevent degradation, increase stability or uptake. In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end) of the MiHA peptide is modified (e.g., for protection against degradation), for example by covalent attachment of a moiety/chemical group (Z1). Z1 may be a straight chained or branched alkyl group of one to eight carbons, or an acyl group (R—CO—), wherein R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar—CO—), wherein Ar is an aryl group. In an embodiment, the acyl group is a C1-C16 or C3-C16 acyl group (linear or branched, saturated or unsaturated), in a further embodiment, a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), for example an acetyl group (CH3—CO—, Ac). In an embodiment, Z1 is absent.
The carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the MiHA peptide) of the MiHA peptide may be modified (e.g., for protection against degradation), for example by amidation (replacement of the OH group by a NH2 group), thus in such a case Z2 is a NH2 group. In an embodiment, Z2 may be an hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of one to ten carbons such as methyl amine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, napthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol or CH2OH. In an embodiment, Z2 is absent.
In an embodiment, the MiHA peptide comprises one of sequences Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 set forth in Table I. In an embodiment, the MiHA peptide consists of one of sequences No. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 set forth in Table I, i.e. wherein Z1 and Z2 are absent.
The MiHA peptides of the invention may be produced by expression in a host cell comprising a nucleic acid encoding the MiHA peptides (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by manual and/or automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing “T-boc” or “Fmoc” procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the MiHA peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Left. 37: 933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45: 209-216, 1995; Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31: 322-334, 1988). Other methods useful for synthesizing the MiHA peptides are described in Nakagawa et al., J. Am. Chem. Soc. 107: 7087-7092, 1985. In an embodiment, the MiHA peptide of the formula I or Ia is chemically synthesized (synthetic peptide).
Accordingly, in another aspect, the invention further provides a nucleic acid (isolated) encoding the above-mentioned MiHA peptides or a MiHA precursor-peptide. In an embodiment, the nucleic acid comprises from about 21 nucleotides to about 45 nucleotides, from about 24 to about 45 nucleotides, for example 24, 27, 30, 33, 36, 39, 42 or 45 nucleotides.
“Isolated”, as used herein, refers to a peptide or nucleic molecule separated from other components that are present in the natural environment of the molecule or a naturally occurring source macromolecule (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). “Synthetic”, as used herein, refers to a peptide or nucleic molecule that is not isolated from its natural sources, e.g., which is produced through recombinant technology or using chemical synthesis.
In an embodiment, the above-mentioned MiHA peptide is substantially pure. A compound is “substantially pure” when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, 80% or 85%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components, e.g. components of its source macromolecule. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
A nucleic acid of the invention may be used for recombinant expression of the MiHA peptide of the invention, and may be included in a vector or plasmid, such as a cloning vector or an expression vector, which may be transfected into a host cell. In an embodiment, the invention provides a cloning or expression vector or plasmid comprising a nucleic acid sequence encoding the MiHA peptide of the invention. Alternatively, a nucleic acid encoding a MiHA peptide of the invention may be incorporated into the genome of the host cell. In either case, the host cell expresses the MiHA peptide or protein encoded by the nucleic acid.
The vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding peptides or polypeptides and appropriate transcriptional and translational control/regulatory elements operably linked thereto. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
“Operably linked” refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and/or translational control, of the regulatory sequences. “Regulatory/control region” or “regulatory/control sequence”, as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid. Thus the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like.
In an embodiment, the MiHA peptide is in solution. In another embodiment, the MiHA peptide is in solid form, e.g., lyophilized.
In another aspect, the present invention provides a MHC class I molecule comprising (i.e. presenting or bound to) a MiHA peptide. In an embodiment, the MHC class I molecule is a HLA-A2 molecule, in a further embodiment a HLA-A*02:01 molecule. In another embodiment, the MHC class I molecule is a HLA-B44 molecule, in a further embodiment a HLA-B*44:03 molecule. In an embodiment, the MiHA peptide is non-covalently bound to the MHC class I molecule (i.e., the MiHA peptide is loaded into, or non-covalently bound to the peptide binding groove/pocket of the MHC class I molecule). In another embodiment, the MiHA peptide is covalently attached/bound to the MHC class I molecule (alpha chain). In such a construct, the MiHA peptide and the MHC class I molecule (alpha chain) are produced as a synthetic fusion protein, typically with a short (e.g., 5 to 20 residues, preferably about 8-12, e.g., 10) flexible linker or spacer (e.g., a polyglycine linker). In another aspect, the invention provides a nucleic acid encoding a fusion protein comprising a MiHA peptide defined above fused to a MHC class I molecule (alpha chain). In an embodiment, the MHC class I molecule (alpha chain)—peptide complex is multimerized. Accordingly, in another aspect, the present invention provides a multimer of MHC class I molecule loaded (covalently or not) with the above-mentioned MiHA peptide. Such multimers may be attached to a tag, for example a fluorescent tag, which allows the detection of the multimers. A great number of strategies have been developed for the production of MHC multimers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Thus, in another aspect, the present invention provides a method for detecting or purifying (isolating, enriching) CD8+ T lymphocytes specific for a MiHA peptide defined above, the method comprising contacting a cell population with a multimer of MHC class I molecule loaded (covalently or not) with the MiHA peptide; and detecting or isolating the CD8+ T lymphocytes bound by the MHC class I multimers. CD8+ T lymphocytes bound by the MHC class I multimers may be isolated using known methods, for example fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).
In yet another aspect, the present invention provides a cell (e.g., a host cell), in an embodiment an isolated cell, comprising the above-mentioned nucleic acid, vector or plasmid of the invention, i.e. a nucleic acid or vector encoding one or more MiHA peptides.
In another aspect, the present invention provides a cell expressing at its surface a MHC class I molecule (e.g., a HLA-A2 or HLA-B44 allele molecule) bound to or presenting a MiHA peptide according to the invention. In one embodiment, the host cell is a primary cell, a cell line or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC).
Nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.
Cells such as APCs can be loaded with one or more MiHA peptides using a variety of methods known in the art. As used herein “loading a cell” with a MiHA peptide means that RNA or DNA encoding the MiHA peptide, or the MiHA peptide, is transfected into the cells or alternatively that the APC is transformed with a nucleic acid encoding the MiHA peptide. The cell can also be loaded by contacting the cell with exogenous MiHA peptides that can bind directly to MHC class I molecule present at the cell surface (e.g., peptide-pulsed cells). The MiHA peptides may also be fused to a domain or motif that facilitates its presentation by MHC class I molecules, for example to an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence (see Wang et al., Eur J Immunol. 2004 December; 34(12):3582-94).
In another aspect, the present invention provides a composition or peptide combination comprising any one of, or any combination of, the MiHA peptides defined above (or a nucleic acid encoding said peptide(s)). In an embodiment, the composition comprises any combination of the MiHA peptides defined above (e.g., any combination of MiHAs Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 set forth in Table I), or a combination of nucleic acids encoding said MiHA peptides). For example, the composition may comprise a first MiHA peptide which correspond to MiHA No. 1 and a second MiHA peptide that corresponds to MiHA No. 24. Compositions comprising any combination/sub-combination of the MiHA peptides defined above are encompassed by the present invention. In another embodiment, the combination may comprise one or more known MiHAs, such as the known MiHAs disclosed herein (see, e.g., Tables III and V). In an embodiment, the composition or peptide combination comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 MiHA peptides, wherein at least one of said MiHA peptide comprising the MiHAs Nos. 1-93
In a further embodiment, a MHC class I molecule (HLA-A2 or HLA-B44) that presents a MiHA peptide is expressed at the surface of a cell, e.g., an APC. In an embodiment, the invention provides an APC loaded with one or more MiHA peptides bound to MHC class I molecules. In yet a further embodiment, the invention provides an isolated MHC class I/MiHA peptide complex.
Thus, in another aspect, the present invention provides a composition comprising any one of, or any combination of, the MiHA peptides defined above and a cell expressing a MHC class I molecule (HLA-A2 or HLA-B44). APC for use in the present invention are not limited to a particular type of cell and include professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages and B cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by CD8+ T lymphocytes. For example, an APC can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) the MiHA peptides, either in vitro, ex vivo or in vivo. APC can also be activated to present a MiHA peptide in vivo where one or more of the MiHA peptides of the invention are administered to a subject and APCs that present a MiHA peptide are induced in the body of the subject. The phrase “inducing an APC” or “stimulating an APC” includes contacting or loading a cell with one or more MiHA peptides, or nucleic acids encoding the MiHA peptides such that the MiHA peptides are presented at its surface by MHC class I molecules (e.g., HLA-A2 or HLA-B44). As noted above, according to the present invention, the MiHA peptides may be loaded indirectly for example using longer peptides/polypeptides comprising the sequence of the MiHAs (including the native protein), which is then processed (e.g., by proteases) inside the APCs to generate the MiHA peptide/MHC class I complexes at the surface of the cells.
After loading APCs with MiHA peptides and allowing the APCs to present the MiHA peptides, the APCs can be administered to a subject as a vaccine. For example, the ex vivo administration can include the steps of:
(a) collecting APCs from a first subject, (b) contacting/loading the APCs of step (a) with a MiHA peptide to form MHC class I/MiHA peptide complexes at the surface of the APCs; and (c) administering the peptide-loaded APCs to a second subject in need for treatment.
The first subject and the second subject can be the same individual (e.g., autologous vaccine), or may be different individuals (e.g., allogeneic vaccine). Alternatively, according to the present invention, use of a MiHA peptide of the present invention for manufacturing a pharmaceutical composition for inducing antigen-presenting cells is provided. In addition, the present invention provides a method or process for manufacturing a pharmaceutical composition for inducing antigen-presenting cells, wherein the method or the process includes the step of admixing or formulating the MiHA peptide with a pharmaceutically acceptable carrier.
Cells such as APCs expressing a MHC class I molecule (HLA-A2 or HLA-B44) loaded with any one of, or any combination of, the MiHA peptides defined above, may be used for stimulating/amplifying CD8+ T lymphocytes, for example autologous CD8+ T lymphocytes. Accordingly, in another aspect, the present invention provides a composition comprising any one of, or any combination of, the MiHA peptides defined above (or a nucleic acid or vector encoding same); a cell expressing a MHC class I molecule (HLA-A2 or HLA-B44) and a T lymphocyte, more specifically a CD8+ T lymphocyte (e.g., a population of cells comprising CD8+ T lymphocytes).
In an embodiment, the composition further comprises a buffer, an excipient, a carrier, a diluent and/or a medium (e.g., a culture medium). In a further embodiment, the buffer, excipient, carrier, diluent and/or medium is/are pharmaceutically acceptable buffer(s), excipient(s), carrier(s), diluent(s) and/or medium (media). As used herein “pharmaceutically acceptable buffer, excipient, carrier, diluent and/or medium” includes any and all solvents, buffers, binders, lubricants, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like that are physiologically compatible, do not interfere with effectiveness of the biological activity of the active ingredient(s) and that are not toxic to the subject. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound (peptides, cells), use thereof in the compositions of the invention is contemplated. In an embodiment, the buffer, excipient, carrier and/or medium is a non-naturally occurring buffer, excipient, carrier and/or medium.
In one embodiment, the MiHA peptides of the invention are used as a vaccine.
In another aspect, the present invention provides an immunogenic composition comprising one of more of the any one of, or any combination of, the MiHA peptides defined above (or a nucleic acid encoding said peptide(s)), and a buffer, an excipient, a carrier, a diluent and/or a medium.
For compositions comprising cells (e.g., T lymphocytes), the composition comprises a suitable medium that allows the maintenance of viable cells. Representative examples of such media include saline solution, Earl's Balanced Salt Solution (Life Technologies®) or PlasmaLyte® (Baxter International®).
In an embodiment, the composition is an “immunogenic composition” or “vaccine”. The term “Immunogenic composition” or “vaccine” as used herein refers to a composition or formulation comprising one or more MiHA peptides or vaccine vector and which is capable of inducing an immune response against the one or more MiHA peptides present therein when administered to a subject. Vaccination methods for inducing an immune response in a mammal comprise use of a vaccine or vaccine vector to be administered by any conventional route known in the vaccine field, e.g., via a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g., via a transdermal delivery system such as a patch).
In an embodiment, the MiHA peptide is conjugated to a carrier protein (conjugate vaccine) to increase the immunogenicity of the MiHA peptide. The present invention thus provides a composition (conjugate) comprising a MiHA peptide and a carrier protein. For example, the MiHA peptide may be conjugated to a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012; 114:177-201) or polymers/dendrimers (see, e.g., Liu et al., Biomacromolecules. 2013 Aug. 12; 14(8):2798-806).
In an embodiment, the immunogenic composition or vaccine further comprises an adjuvant. “Adjuvant” refers to a substance which, when added to an immunogenic agent such as an antigen (MiHA peptides and/or cells according to the present invention), nonspecifically enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. Examples of adjuvants currently used in the field of vaccines include (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and/or (6) inert vehicles, such as gold particles, and the like.
In an embodiment, the MiHA peptide(s) or composition comprising same is in lyophilized form. In another embodiment, the MiHA peptide(s) is/are in a liquid composition. In a further embodiment, the MiHA peptide(s) is/are at a concentration of about 0.01 μg/mL to about 100 μg/mL in the composition. In further embodiments, the MiHA peptide(s) is/are at a concentration of about 0.2 μg/mL to about 50 μg/mL, about 0.5 μg/mL to about 10, 20, 30, 40 or 50 μg/mL, about 1 μg/mL to about 10 μg/mL, or about 2 μg/mL, in the composition.
As noted above, cells such as APCs that express a MHC class I molecule (HLA-A2 or HLA-B44) loaded with or bound to any one of, or any combination of, the MiHA peptides defined above, may be used for stimulating/amplifying CD8+ T lymphocytes in vivo or ex vivo.
Accordingly, in another aspect, the present invention provides T cell receptor (TCR) molecules capable of interacting with or binding the above-mentioned MHC class I molecule/MiHA peptide complex, and nucleic acid molecules encoding such TCR molecules, and vectors comprising such nucleic acid molecules. A TCR according to the present invention is capable of specifically interacting with or binding a MiHA peptide loaded on, or presented by, a MHC class I molecule (HLA-A2 or HLA-B44), preferably at the surface of a living cell in vitro or in vivo. A TCR and in particular nucleic acids encoding a TCR of the invention may for instance be applied to genetically transform/modify T lymphocytes (e.g., CD8+ T lymphocytes) or other types of lymphocytes generating new T lymphocyte clones that specifically recognizing a MHC class I MiHA peptide complex. In a particular embodiment, T lymphocytes (e.g., CD8+ T lymphocytes) obtained from a patient are transformed to express one or more TCRs that recognize MiHA peptide and the transformed cells are administered to the patient (autologous cell transfusion).
In another embodiment, the invention provides a T lymphocyte e.g., a CD8+ T lymphocyte transformed/transfected by a vector or plasmid encoding a MiHA peptide-specific TCR. In a further embodiment the invention provides a method of treating a patient with autologous or allogenic cells transformed with a MiHA-specific TCR. In yet a further embodiment the use of a MiHA specific TCR in the manufacture of autologous or allogenic cells for treating of cancer is provided.
In some embodiments patients treated with the therapeutic compositions of the invention are treated prior to or following treatment with allogenic stem cell transplant (ASCL), allogenic lymphocyte infusion or autologous lymphocyte infusion. Therapeutic compositions of the invention include: allogenic T lymphocytes (e.g., CD8+ T lymphocyte) activated ex vivo against a MiHA peptide; allogenic or autologous APC vaccines loaded with a MiHA peptide; MiHA peptide vaccines and allogenic or autologous T lymphocytes (e.g., CD8+ T lymphocyte) or lymphocytes transformed with a MiHA-specific TCR.
The method to provide T lymphocyte clones capable of recognizing an MiHA peptide according to the invention may be generated for and can be specifically targeted to tumor cells expressing the MiHA in a subject (e.g., graft recipient), for example an ASCT and/or donor lymphocyte infusion (DLI) recipient. Hence the invention provides a CD8+ T lymphocyte encoding and expressing a T cell receptor capable of specifically recognizing or binding a MiHA peptide/MHC class I molecule complex. Said T lymphocyte (e.g., CD8+ T lymphocyte) may be a recombinant (engineered) or a naturally selected T lymphocyte. This specification thus provides at least two methods for producing CD8+ T lymphocytes of the invention, comprising the step of bringing undifferentiated lymphocytes into contact with a MiHA peptide/MHC class I molecule complex (typically expressed at the surface of cells, such as APCs) under conditions conducive of triggering T cell activation and expansion, which may be done in vitro or in vivo (i.e. in a patient administered with a APC vaccine wherein the APC is loaded with a MiHA peptide or in a patient treated with a MiHA peptide vaccine). Alternatively, MiHA-specific or targeted T lymphocytes may be produced/generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding a TCR (more specifically the alpha and beta chains) that specifically binds to a MHC class I molecule/MiHA complex (i.e. engineered or recombinant CD8+ T lymphocytes). Nucleic acids encoding a MiHA-specific TCR of the invention, may be obtained using methods known in the art from a T lymphocyte activated against a MiHA peptide ex vivo (e.g., with an APC loaded with a MiHA peptide); or from an individual exhibiting an immune response against peptide/MHC molecule complex. MiHA-specific TCRs of the invention may be recombinantly expressed in a host cell and/or a host lymphocyte obtained from a graft recipient or graft donor, and optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). The nucleic acid(s) (transgene(s)) encoding the TCR alpha and beta chains may be introduced into a T cells (e.g., from a subject to be treated or another individual) using any suitable methods such as transfection (e.g., electroporation) or transduction (e.g., using viral vector). The engineered CD8+ T lymphocytes expressing a TCR specific for a MiHA may be expanded in vitro using well known culturing methods.
The present invention provides isolated CD8+ T lymphocytes that are specifically induced, activated and/or amplified (expanded) by a MiHA peptide (i.e., a MiHA peptide bound to MHC class I molecules expressed at the surface of cell). The present invention also provides a composition comprising CD8+ T lymphocytes capable of recognizing an MiHA peptide according to the invention (i.e., a MiHA peptide bound to MHC class I molecules) and said MiHA peptide.
In another aspect, the present invention provides a cell population or cell culture (e.g., a CD8+ T lymphocyte population) enriched in CD8+ T lymphocytes that specifically recognize a MHC class I molecule/MiHA peptide complex as described herein. Such enriched population may be obtained by performing an ex vivo expansion of specific T lymphocytes using cells such as APCs that express MHC class I molecules loaded with e.g. presenting) one or more of the MiHA peptides disclosed herein. “Enriched” as used herein means that the proportion of MiHA-specific CD8+ T lymphocytes in the population is significantly higher relative to a native population of cells, i.e. which has not been subjected to a step of ex vivo-expansion of specific T lymphocytes. In a further embodiment, the proportion of MiHA-specific CD8+ T lymphocytes in the cell population is at least about 0.5%, for example at least about 1%, 1.5%, 2% or 3%. In some embodiments, the proportion of MiHA-specific CD8+ T lymphocytes in the cell population is about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 5%, about 0.5 to about 4%, about 0.5 to about 3%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5% or about 3% to about 4%. Such cell population or culture (e.g., a CD8+ T lymphocyte population) enriched in CD8+ T lymphocytes that specifically recognizes a MHC class I molecule/peptide (MiHA) complex of interest may be used in MiHA-based cancer immunotherapy, as detailed below.
In some embodiments, the population of MiHA-specific CD8+ T lymphocytes is further enriched, for example using affinity-based systems such as multimers of MHC class I molecule loaded (covalently or not) with the MiHA peptide defined above. Thus, the present invention provides a purified or isolated population of MiHA-specific CD8+ T lymphocytes, e.g., in which the proportion of MiHA-specific CD8+ T lymphocytes is at least 50%, 60%, 70%, 80%, 85%, 90% or 95%.
The MiHA peptide sequences identified herein may be used for the production of synthetic peptides to be used i) for in vitro priming and expansion of MiHA-specific T cells to be injected into transplant (AHCT) recipients and/or ii) as vaccines to boost the graft-vs.-tumor effect (GvTE) in recipients of MiHA-specific T cells, subsequent to the transplantation.
The potential impact of the therapeutic methods provided by the present invention, MiHA-targeted cancer immunotherapy is significant. For hematologic cancers (e.g., leukemia, lymphoma and myeloma), the use of anti-MiHA T cells may replace conventional AHCT by providing superior anti-tumor activity without causing GvHD. It may benefit many patients with hematologic malignancy who cannot be treated by conventional AHCT because their risk/reward (GvHD/GVT) ratio is too high. Finally, since studies in mice have shown that MiHA-targeted immunotherapy may be effective for treatment of solid tumors, MiHA-based cancer immunotherapy may be used for MiHA-targeted therapy of non-hematologic cancers, such as solid cancers.
In embodiment, the cancer is leukemia including but not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) chronic myeloid leukemia (CML), Hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), Large granular lymphocytic leukemia or Adult T-cell leukemia. In another embodiment, the cancer is lymphoma including but not limited to Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Burkitt's lymphoma, Precursor T-cell leukemia/lymphoma, Follicular lymphoma, Diffuse large B cell lymphoma, Mantle cell lymphoma, B-cell chronic lymphocytic leukemia/lymphoma or MALT lymphoma. In a further embodiment, the cancer is a myeloma (multiple myeloma) including but not limited to plasma cell myeloma, myelomatosis, and Kahler's disease.
In another aspect, the present invention provides the use of a MiHA peptide of the present invention as a vaccine for treating cancer in a subject. In an embodiment, the subject is a recipient of MiHA-specific CD8+ T lymphocytes.
Accordingly, in another aspect, the present invention provides a method of treating cancer (e.g., of reducing the number of tumor cells, killing tumor cells), said method comprising administering (infusing) to a subject in need thereof an effective amount of CD8+ T lymphocytes recognizing (i.e. expressing a TCR that binds) a MHC class I molecule/MiHA peptide complex (expressed at the surface of a cell such as an APC). In an embodiment, the method further comprises administering an effective amount of the MiHA peptide, and/or a cell (e.g., an APC such as a dendritic cell) expressing MHC class I molecule loaded with the MiHA peptide, to said subject after administration/infusion of said CD8+ T lymphocytes. In yet a further embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of a dendritic cell loaded with one or more MiHA peptides. In yet a further embodiment the method comprises administering to a patient in need thereof a therapeutically effective amount of a allogenic or autologous cell that expresses a recombinant TCR that binds to a MiHA peptide presented by a MHC class I molecule.
In another aspect, the present invention provides the use of CD8+ T lymphocytes that recognize a MHC class I molecule loaded with (presenting) a MiHA peptide for treating cancer (e.g., of reducing the number of tumor cells, killing tumor cells) in a subject. In another aspect, the present invention provides the use of CD8+ T lymphocytes that recognize a MHC class I molecule loaded with a MiHA peptide for the preparation/manufacture of a medicament for treating cancer (e.g., fir reducing the number of tumor cells, killing tumor cells) in a subject.
In another aspect, the present invention provides CD8+ T lymphocytes that recognize a MHC class I molecule loaded with (presenting) a MiHA peptide for use in the treatment of cancer (e.g., for reducing the number of tumor cells, killing tumor cells) in a subject.
In a further embodiment, the use further comprises the use of an effective amount of a MiHA peptide, and/or of a cell (e.g., an APC) that expresses a MHC class I molecule loaded with (presenting) a peptide of formula I, after the use of said MiHA-specific CD8+ T lymphocytes.
In an embodiment, the subject infused or treated with MiHA-specific CD8 T-cells has received prior treatment with AHCT or donor lymphocyte infusions (i.e. lymphocytes, including T-cells, that have not been activated in vitro against a MiHA peptide presented by a MHC class I molecule. In a further embodiment, the cancer is a hematologic cancer, e.g., leukemia, lymphoma and myeloma. In an embodiment, the cancer is leukemia.
Allogenic therapeutic cells of the invention express a TCR that recognizes a MiHA peptide that is presented by a patient's (recipient's) tumor cells but not presented by cells of the donor. The invention provides a method of selecting an effective therapeutic composition for a patient having hematological cancer comprising: (a) obtaining a biological sample from the patient; (b) determining the presence or absence of one or more SNPs selected from Table II; (c) determining the expression of RNA or protein products corresponding to one or more of the SNPs provided in Table II in a tumor sample from the patient. For treatment with allogenic cells: (a) a donor that does not express a genetic variant, corresponding to a MiHA peptide (i.e. those provided in Table II herein) presented by MHC class I molecules expressed by the recipient's cancer cells is selected (b) lymphocytes are obtained from the donor and (c) CD8+ T lymphocytes specific for the presented MiHA peptide are prepared using the methods provided herein and administered to the patient. Alternatively allogenic cells obtained from the selected donor, one that does not express the MiHA of interest, can be genetically transformed to express a TCR against the MiHA of interest and administered to the patient.
For treatment with autologous cells, an autologous T lymphocyte expressing a TCR that recognizes a MiHA presented by MHC class I molecules present on the cell surface of a patient's cancer cells is administered. The invention provides a method of selecting a T lymphocyte therapy for a patient comprising: (a) obtaining a biological sample from the patient; (b) determining the presence or absence of one or more SNPs selected from Table II; (c) determining the expression of RNA or protein products corresponding to one or more of the SNPs provided in Table II in a tumor sample from the patient.
To determine which variant of a given MiHA that should be used in the treatment of a subject (e.g., using MiHA No. 1 as an example, to determine which of SED ID NO: 80 or 81 should be used), the allelic variant expressed by the subject should be first determined. The amino acid substitutions in the proteins as well as the nucleotide substitutions in the transcripts corresponding to the novel MiHAs described herein (Table II) may be easily identified by the skilled person, for example using the information provided in public databases. For example, Table II includes the reference/identification No. for MiHAs in the dbSNP database, which provides detailed information concerning the variations at the genomic, transcript and protein levels. Based on this information, the determination of the variant (polymorphism or single nucleotide polymorphism (SNP)) expressed by the subject may be readily performed at the nucleic acid and/or protein level on a sample by a number of methods which are known in the art. Table II also includes the reference ID in the Ensembl database for the genes from which the MiHA peptides are derived.
Examples of suitable methods for detecting alterations at the nucleic acid level include sequencing the relevant portion (comprising the variation) of the nucleic acid of interest (e.g., a mRNA, cDNA or genomic DNA encoding the MiHAs), hybridization of a nucleic acid probe capable of specifically hybridizing to a nucleic acid of interest comprising the polymorphism (the first allele) and not to (or to a lesser extent to) a corresponding nucleic acid that do not comprise the polymorphism (the second allele) (under comparable hybridization conditions, such as stringent hybridization conditions), or vice-versa; restriction fragment length polymorphism analysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR); amplification of a nucleic acid fragment using a primer specific for one of the allele, wherein the primer produces an amplified product if the allele is present and does not produce the same amplified product when the other allele is used as a template for amplification (e.g., allele-specific PCR). Other methods include in situ hybridization analyses and single-stranded conformational polymorphism analyses. Further, nucleic acids of interest may be amplified using known methods (e.g., polymerase chain reaction [PCR]) prior to or in conjunction with the detection methods noted herein. The design of various primers for such amplification is known in the art. The nucleic acid (mRNA) may also be reverse transcribed into cDNA prior to analysis.
Examples of suitable methods for detecting alterations/polymorphisms at the polypeptide level include sequencing of the relevant portion (comprising the variation) of the polypeptide of interest, digestion of the polypeptide followed by mass spectrometry or HPLC analysis of the peptide fragments, wherein the variation/polymorphism of the polypeptide of interest results in an altered mass spectrometry or HPLC spectrum; and immunodetection using an immunological reagent (e.g., an antibody, a ligand) which exhibits altered immunoreactivity with a polypeptide comprising the alteration (first allele) relative to a corresponding native polypeptide not comprising the alteration (second allele), for example by targeting an epitope comprising the amino acid variation. Immunodetection can measure the amount of binding between a polypeptide molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody. In addition, other high affinity ligands may be used. Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999).
All these detection techniques may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000).
In one embodiment the invention provides a method of selecting an effective therapeutic composition for a patient comprising: (a) isolating MHC class I presented peptides from hematologic cancer cells from the patient; and (b) identifying the presence or absence of one or more MiHA peptides depicted in Table I among said MHC class I presented peptides. In a further embodiment, the identification of the presence or absence of the one or more MiHA peptides depicted in Table I is performed by mass spectrometry and/or using an antibody detection reagent that is selective for the one or more MiHA peptides. Detecting or identifying MiHA peptides using mass spectrometry can be performed using methods known in the art such as those described in PCT publication No. WO2014/026277. Mass spectrometry (MS) sequencing of MiHA peptides presented by MHC class I molecules, which have been isolated from a sample of cancer cells, involves comparing a MS spectra obtained for an isolated and digested peptide to spectra computed in silico for a MiHA peptide.
Therapeutic allogenic T lymphocytes of the present invention, for treating a patient with cancer, target MHC class I molecules presenting one or more MiHA peptides that is/are expressed by cancer cells in the patient but not expressed by the donor's cells. As such, selecting an appropriate donor for generating allogenic T lymphocytes of the invention involves genotyping candidate donors for the presence or absence of one or more single nucleotide polymorphisms provided in Table II.
In one embodiment the invention provides a method of selecting an effective immunotherapy treatment (i.e. MHC class I molecule/MiHA peptide complex target) for a patient with cancer comprising: determining the presence of MiHA peptides presented by MHC class I molecules in tumor cells from the patient.
In another embodiment the invention provides a method of screening candidate allogenic cell donors for a patient comprising determining the presence or absence of one or more SNPs selected from those provided in Table II in a biological sample from the donor. In an embodiment, the presence or absence of a SNP corresponding to a MiHA peptide known to be presented by MHC class I molecule in cancer cells obtained from a patient is determined in candidate donors. In a further embodiment, biological samples obtained from candidate allogenic donors are genotyped to determine the presence or absence of one or more SNPs known to be carried by a patient, wherein the SNPs detected are selected from those provided in Table II.
In a further embodiment the invention provides a genotyping system comprising a plurality of oligonucleotide probes conjugated to a solid surface for detection of a plurality of SNPs selected from Table II.
For example, to determine which variant of MiHA No. 1 (SEQ ID Nos. 80 or 81) should be used in the treatment of a subject, it should be determined on a sample from the subject using any suitable method (sequencing, etc.) whether (i) a transcript from the ANKRD13A gene comprises a T or C at a position corresponding to position 1680 of Ensembl Transcript ID No. ENST0000261739 (ENSG00000076513); (ii) the nucleotide corresponding to position 110036265 of chromosome 12 in human genome assembly GRCh38 is C or T; and/or (iii) an ANKRD13A polypeptide comprises a leucine or proline residue at a position corresponding to position 505 of the polypeptide encoded by Ensembl Transcript ID No. ENST0000261739 (ENSG00000076513). If (i) the transcript from the ANKRD13A gene comprises a T at a position corresponding to position 1680 of Ensembl Transcript ID No. ENST0000261739; (ii) the nucleotide corresponding to position 110036265 of chromosome 12 in human genome assembly GRCh38 is T; and/or (iii) the ANKRD13A polypeptide comprises a leucine residue at a position corresponding to position 505 of the polypeptide encoded by Ensembl Transcript ID No. ENST0000261739, MiHA variant of SEQ ID No. 80 (SLLESSRSQEL) should be used. Alternatively, if (i) the transcript from the ANKRD13A gene comprises a C at a position corresponding to position 1680 of Ensembl Transcript ID No. ENST0000261739; (ii) the nucleotide corresponding to position 110036265 of chromosome 12 in human genome assembly GRCh38 is C; and/or (iii) the ANKRD13A polypeptide comprises a proline residue at a position corresponding to position 505 of the polypeptide encoded by Ensembl Transcript ID No. ENST0000261739, MiHA variant of SEQ ID No. 81 (SLLESSRSQEP) should be used. The same approach may be applied to determine which variant of any of MiHAs Nos. 2-5, 7-25, 27-30 and 32-84 should be used in a given subject.
MiHAs Nos. 34 and 35 may only be used in male subjects (since the encoding gene is located on chromosome Y, the MiHA is only expressed in male subjects).
In an embodiment, the above-mentioned CD8+ T lymphocytes are in vitro or ex vivo expanded CD8+ T lymphocytes, as described above. Expanded CD8+ T lymphocytes may be obtained by culturing primary CD8+ T lymphocytes (from an allogenic donor) under conditions permitting the proliferation (amplification) and/or differentiation of the CD8+ T lymphocytes. Such conditions typically include contacting the CD8+ T lymphocytes with cells, such as APCs, expressing at their surface the MiHA peptide/MHC complexes of interest, in the presence of a suitable medium (medium for hematopoietic/lymphoid cells, e.g., X-VIVO™15 and AIM-V®) growth factors and/or cytokines such as IL-2, IL-7 and/or IL-15 (see, e.g., Montes et al., Clin Exp Immunol. 2005 November; 142(2):292-302). Such expanded CD8+ T lymphocytes are then administered to the recipient, for example through intravenous infusion. Methods and conditions for amplifying and preparing antigen-specific CD8+ T lymphocytes for adoptive immunotherapy are disclosed, for example, in DiGiusto et al., Cytotherapy 2007; 9(7): 613-629 and Bollard et al., Cytotherapy. 2011 May; 13(5): 518-522). Standard Operating procedures (SOPs) for amplifying antigen-specific CD8+ T lymphocytes are available from the Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children's Hospital, The Methodist Hospital, Houston, Tex., USA (see Sili et al., Cytotherapy. 2012 January; 14(1): 7-11, Supplementary Material).
In an embodiment, the subject (recipient) is an allogeneic stem cell transplantation (ASCT) or donor lymphocyte infusion (DLI) recipient.
In another aspect, the present invention provides a method of expanding CD8+ T lymphocytes (e.g., for adoptive T-cell immunotherapy), said method comprising (a), culturing CD8+ T lymphocytes from a first individual not expressing a variant of a MiHA peptide in the presence of cells expressing a MHC class I molecule of the HLA-A2 and/or HLA-B44 allele loaded with said variant of the MiHA peptide, under conditions suitable for CD8+ T lymphocyte expansion.
In another aspect, the invention provides a method of producing/manufacturing cells for cellular immunotherapy comprising: culturing human lymphocytes in the presence of APC comprising a MiHA peptide presented by a MHC class I molecule, wherein the MHC class I molecule is of the HLA-A2 or ALA-B44 subtype. The human T lymphocyte used in this method is an allogenic cell i.e. a cell obtained from a donor being manufactured for treating a recipient with an allogenic cell.
In another aspect, the invention provides a method of producing/manufacturing cells for cellular immunotherapy comprising: (a) obtaining lymphocytes (e.g., T lymphocytes) from a cultured cell line and (b) culturing the cells in the presence of APC comprising a MHC class I molecule/MiHA peptide complex wherein the MHC class I molecule is a HLA-A2 or ALA-B44 subtype. The human T lymphocyte used in the method is preferably an allogenic cell i.e. a cell obtained from a donor being manufacture for treating a recipient with an allogenic cell.
In a further embodiment, the invention provides a method of producing/manufacturing cells for cellular immunotherapy comprising: (a) obtaining cells from a donor, e.g., a patient having a hematopoietic cancer (e.g., leukemia) or a healthy individual, for example by leukapheresis, and (b) transforming the cells with a recombinant TCR that binds to a MHC class I molecule/MiHA peptide complex.
In a further embodiment, the invention provides a method of manufacturing cells for cellular immunotherapy comprising transforming a human cell line with a recombinant TCR that binds with to a MHC class I molecule/MiHA peptide complex as defined herein.
In another aspect, the present invention provides a method of expanding CD8+ T lymphocytes for adoptive T-cell immunotherapy, said method comprising (a) determining which variant of any of MiHA Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 is expressed by a subject (recipient), culturing CD8+ T lymphocytes from a candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A2 and/or HLA-B44 allele loaded with the MiHA variant expressed by the subject, under conditions suitable for CD8+ T lymphocyte expansion, wherein said candidate donor does not express the MiHA variant (expressed by the subject (recipient)).
In another aspect, the invention provides a method of selecting a therapeutic approach for a patient having leukemia: (a) detecting the presence of a MiHA peptide presented by a MHC class I molecule expressed in leukemic cells obtained from the patient; and (b) determining the presence or absence of a SNP corresponding to the MiHA peptide detected in step (a), as indicated in Table II, in biological samples obtained from candidate donors.
In another aspect, the invention provides a method of preparing a therapeutic composition for a patient having leukemia: (a) detecting the presence of a MiHA peptide presented by a MHC class I molecule expressed in leukemic cells obtained from the patient; (b) obtaining lymphocytes from the patient by leukapheresis, and (c) transforming said lymphocytes with a TCR that recognizes the presented MiHA peptide detected in step (a).
In another aspect, the invention provides a method of preparing a therapeutic composition for a patient having leukemia: (a) genotyping the patient to determine the presence of a plurality of SNPs selected from Table II; (b) determining the presence of one of the SNPs in the patient (c) obtaining cells from the patient by leukapheresis, and (d) incubating said cells with a APC expressing a MHC class I molecule/MiHA peptide complex, comprising a MiHA peptide that contains the polymorphism encoded by the SNP present in said patient.
Again using MiHA No. 1 (SEQ ID NO: 79) as a representative example to illustrate the method, if it is determined that in a sample from the subject: (i) the transcript from the ANKRD13A gene comprises a T at a position corresponding to position 1680 of Ensembl Transcript ID No. ENST0000261739; (ii) the nucleotide corresponding to position 110036265 of chromosome 12 in human genome assembly GRCh38 is T; and/or (iii) the ANKRD13A polypeptide comprises a leucine residue at a position corresponding to position 505 of the polypeptide encoded by Ensembl Transcript ID No. ENST0000261739, the CD8+ T lymphocytes from the candidate donor are cultured in the presence of cells expressing a MHC class I molecule of the HLA-A2 allele loaded with MiHA variant of SEQ ID No. 80 (SLLESSRSQEL) under conditions suitable for CD8+ T lymphocyte expansion. Alternatively, if it is determined that in a sample from the subject: (i) the transcript from the ANKRD13A gene comprises a C at a position corresponding to position 1680 of Ensembl Transcript ID No. ENST0000261739; (ii) the nucleotide corresponding to position 110036265 of chromosome 12 in human genome assembly GRCh38 is C; and/or (iii) the ANKRD13A polypeptide comprises a proline residue at a position corresponding to position 505 of the polypeptide encoded by Ensembl Transcript ID No. ENST0000261739, the CD8+ T lymphocytes from the candidate donor are cultured in the presence of cells expressing a MHC class I molecule of the HLA-A2 allele loaded with MiHA variant of SEQ ID No. 81 (SLLESSRSQEP) under conditions suitable for CD8+ T lymphocyte expansion. The same approach may be applied to any of MiHAs Nos. 2-93 defined herein.
In an embodiment, the present invention provides a method of treating cancer, said method comprising (i) expanding CD8+ T lymphocytes recognizing a MHC class I molecule loaded with a peptide of formula I for adoptive T-cell immunotherapy according to the method defined above; and (ii) administering (infusing) to a subject in need thereof an effective amount of the expanded CD8+ T lymphocytes. In one embodiment, the method further comprises administering an effective amount of the peptide of formula I, and/or a cell (e.g., an APC) expressing MHC class I molecule loaded with a MiHA peptide of formula I, to said subject after administration/infusion of said CD8+ T lymphocytes.
In embodiment, the above-mentioned cancer comprises tumor cells expressing the genes encoding MiHAs Nos. 1-93, preferably MiHAs 7, 8, 10, 14-15, 20-21, 32, 33, 35, 39-47, 49-50, 66, 70-71, 76, 78, 81, 84 and 90 set forth in Table I.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
The present invention is illustrated in further details by the following non-limiting examples.
The MiHAs were identified according to the method/strategy described in PCT publication No. WO 2014/026277 and (3). A schematic overview of the procedure is depicted in
Cell Culture.
Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 6 female and 7 male volunteers expressing at least one of the following two common alleles HLA-A*02:01 and HLA-B*44:03. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) were derived from PBMCs with Ficoll-Paque™ Plus (Amersham) as previously described (Tosato and Cohen, 2007). Established B-LCLs were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM of HEPES, 2 mM L-glutamine and penicillin-streptomycin (all from Invitrogen).
DNA Extraction.
Genomic DNA was extracted from 5 million B-LCLs using the PureLink™ Genomic DNA Mini Kit (Invitrogen®) according to the manufacturer's instructions. DNA was quantified and quality-assessed using the Taqman® RNase P Detection Reagents Kit (Life Technologies®).
HLA Typing.
High-resolution HLA genotyping was performed using 500 ng of genomic DNA at the Maisonneuve-Rosemont Hospital.
Preparation of Genomic DNA Libraries.
Genomic libraries were constructed from 200 ng of genomic DNA using the Ion AmpliSeg™ Exome RDY Library Preparation Kit (Life Technologies®) following the manufacturer's protocol. This included the following steps: amplification of targets, partial digestion of primer sequences, ligation of Ion Xpress™ barcode adapters to the amplicons, purification of the library using AMPure® XP reagent (Beckman Coulter®) and quantification of the unamplified library by qPCR. Library templates were then prepared and loaded onto Ion Proton™ I chips using the Ion PI™ IC 200 kit and the Ion Chef™ System.
Exome Sequencing and Variant Calling.
Two exome libraries were sequenced per chip on an Ion Proton™ Sequencer using the default parameters of AmpliSeg™ exome libraries. Variant calling was done using the Torrent Variant Caller plugin with the “germ Line Proton-Low Stringency” parameter of the Ion reporter Software.
RNA Extraction.
Total RNA was isolated from 5 million B-LCLs using TRizol RNA reagent (Life Technologies®) including DNase I treatment (Qiagen®) according to the manufacturer's instructions. Total RNA was quantified using the NanoDrop™ 2000 (Thermo Scientific®) and RNA quality was assessed with the 2100 Bioanalyzer™ (Agilent Technologies®).
Preparation of Transcriptome Libraries.
Libraries were generated from 1 μg of total RNA using the TruSeq™ RNA Sample Prep Kit (v2) (RS-930-1021, Illumina®) following the manufacturer's protocol. Briefly, poly-A mRNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. Reverse transcription (RT) of the first strand was done using random primers and SuperScript™ II (InvitroGene®). A second round of RT was also done to generate a double-stranded cDNA, which was then purified using Agencourt AMpure™ XP PCR purification system (Beckman Coulter®). End repair of fragmented cDNA, adenylation of the 3′ ends and ligation of adaptors were done following the manufacturer's protocol. Enrichment of DNA fragments containing adapter molecules on both ends was done using 15 cycles of PCR amplification and the Illumina® PCR mix and primers cocktail.
Whole Transcriptome Sequencing (RNA-Seq).
Paired-end (2×100 bp) sequencing was performed using the Illumina HiSeq2000™ machine running TruSeq™ v3 chemistry. Cluster density was targeted at around 600-800 k clusters/mm2. Two transcriptomes were sequenced per lane (8 lanes per slide). Details of the Illumina sequencing technologies can be found at http://www.illumina.com/applications/detail/sequencing/dna sequencing.ilmn.
Read Mapping.
Sequence data were mapped to the human reference genome (hg19, UCSC) using the Ilumina Casava™ 1.8.1 and the Eland T™ v2 mapping softwares. First, the *.bcl files were converted into compressed FASTQ files, following by demultiplexing of separate multiplexed sequence runs by index. Then, single reads were aligned to the human reference genome including the mitochondrial genome using the multiseed and gapped alignment method. Reads that mapped at 2 or more locations (multireads) were not included in further analyses. An additional alignment was done against splice junctions and contaminants (ribosomal RNA).
Identification of single nucleotide variations in the transcriptome. First, the list of all single nucleotide variations observed between the reference genome (GRCh37.p2, NCBI) and the sequenced transcriptome of each of the individuals was retrieved. This was done using the SNP calling program Casava™ v1.8.2 from Ilumina® (http://support.illumina.com/sequencing/sequencing software/casava.ilmn). Only high confidence single nucleotide variations (Qmax_gt value>20) and that were observed in at least 3 reads (coverage≧3) were considered. SNVs with Qmax_gt value below this threshold were assigned with the reference base instead. This strategy was used to identify single nucleotide variations at the transcript level between each of the individuals and the reference genome.
In Silico Translated Transcriptome.
The sequences containing the identified single nucleotide variations of each individual were further processed. For each sequence, all transcripts reported in Ensembl (http://useast.ensembl.org/info/data/ftp/index.html, Flicek et al., Ensembl 2012, Nucleic Acids Research 2012 40 Database issue:D84-D90) were retrieved and in silico translated into proteins using an in-house software pyGeno version (python package pyGeno 1.1.7, https://pypi.python.org/pypi/pyGeno/1.1.7), Granados et al., 2012 (PMID: 22438248)). The in silico translated transcriptomes included cases in which more than one non-synonymous polymorphism was found for a given position. Considering that most MAPs have a maximum length of 11 amino acids (33 bp), the existence of a heterozygous position could lead to MiHA variants in a window of 21 (66 bp) amino acids centered at each ns-SNP. When a window contained more than one ns-SNP, all possible combinations were translated. The number of combinations affected by one ns-SNP was limited to 10,240 to limit the size of the file. In this way, a list of all possible sequences of at most 11 amino acids affected by ns-SNPs was obtained and included in the individual-specific protein databases, which were further used for the identification of MAPs.
Mass Spectrometry and Peptide Sequencing.
3 to 4 biological replicates of 5-6×108 exponentially growing B-LCLs were prepared from each individual. MHC class I-associated peptides were released by mild acid treatment, pretreated by desalting with an HLB cartridge and filtered with a 3,000 Da cut-off column as previously described (Caron et al. 2011 (PMID: 21952136)). Samples were further processed according to two different methods. In the first method, samples were vacuum dried, resuspended in SCX Reconstitution Solution (Protea®) and separated into six fractions using SCX spintips (Protea®) and an ammonium formate buffer step gradient (50, 75, 100, 300, 600, 1500 mM). Vacuum dried fractions were resuspended in 5% acetonitrile, 0.2% formic acid and analyzed by LC-MS/MS using an Eksigent® LC system coupled to a LTQ-Orbitrap ELITE™ mass spectrometer (Thermo Electron®). Peptides were separated on a custom C18 reversed phase column (pre-column: 0.3 mm i.d.×5 mm, analytical column: 150 μm i.d.×100 mm; Jupiter® C18 3 μm 300 Å) using a flow rate of 600 nL/min and a linear gradient of 5-40% aqueous ACN (0.2% formic acid) in 56 min. Full mass spectra were acquired with the Orbitrap® analyzer operated at a resolving power of 60,000 (at m/z 400). Mass calibration used an internal lock mass (protonated (Si(CH3)2O))6; m/z 445.120029) and mass accuracy of peptide measurements was within 5 ppm. MS/MS spectra were acquired at higher energy collisional dissociation with normalized collision energy of 28. Up to ten precursor ions were accumulated to a target value of 50,000 with a maximum injection time of 100 ms and fragment ions were transferred to the Orbitrap® analyzer operating at a resolution of 60,000 at m/z 400. In the second method, samples were split into two identical technical replicates following the 3,000 Da filtration step and vacuum-dried. One technical replicate was resuspended in 3% acetonitrile, 0.2% formic acid and analyzed by LC-MS/MS using an EASY-nLC® II system coupled to a Q-Exactive™ Plus mass spectrometer (Thermo Scientific®). Peptides were separated on a custom C18 reversed phase column as in the first method, using a flow rate of 600 nl/min and a linear gradient of 3-25% aqueous ACN (0.2% formic acid) in 146 min followed by 25-40% in 5 min. Full mass spectra were acquired with the Orbitrap® analyzer operated at a resolving power of 70,000 (at m/z 400). Mass calibration used an internal lock mass (protonated (Si(CH3)2O))6; m/z 445.120029) and mass accuracy of peptide measurements was within 5 ppm. MS/MS spectra were acquired at higher energy collisional dissociation with normalized collision energy of 25. Up to twelve precursor ions were accumulated to a target value of 1,000,000 with a maximum injection time of 200 ms and fragment ions were transferred to the Orbitrap® analyser operating at a resolution of 17,500 at m/z 400.
MS/MS Sequencing and Peptide Clustering.
Database searches were performed against databases specific to each individual (see ‘in silico-generated proteome and personalized databases’ section) using PEAKS®7 (Bioinformatics Solutions Inc., http://www.bioinfor.com/). Mass tolerances for precursor and fragment ions were set to 5 p.p.m. and 0.02 Da, respectively. Searches were performed without enzyme specificity and with variable modifications for cysteinylation, phosphorylation (Ser, Thr and Tyr), oxidation (Met) and deamidation (Asn, Gln). Raw data files were converted to peptide maps comprising m/z values, charge state, retention time and intensity for all detected ions above a threshold of 30,000 counts. Using in-house software (Proteoprofile) (Granados et al. 2014), peptide maps corresponding to all identified peptide ions were aligned together to correlate their abundances across sample replicates. PEAKS decoy-fusion approach was used to calculate the false discovery rate of quantified unique peptide sequences. The highest scored MS/MS spectra of MHC class I peptides detected in at least one of the individuals were validated manually, using Xcalibur™ software version 2.2 SP1.48 (Thermo Scientific®).
Selection of MiHAs.
Peptides were filtered by their length and those peptides with the canonical MAP length (typically 8-14 mers) were kept. The predicted binding affinity (IC50) of peptides to the allelic products was obtained using NetMHC version 3.4 (http://www.cbs.dtu.dk/services/NetMHC/, Lundegaard et al., 2008 (PMID: 18413329)). Peptides with an IC50 below 5,000 nM were considered as HLA binders.
MiHAs were selected according to the following criteria:
The RNA (cDNA) and DNA sequences encoding MiHAs were manually inspected using the Integrative Genomics Viewer v2.3.25 (The Broad Institute). The UCSC Repeat Masker track was included to discard candidates that corresponded to repetitive regions.
Determination of Allele Frequency.
The minor allele frequency (MAF) of each ns-SNP was obtained from the dbSNP database build 138 (NCBI) and/or the NHLBI Exome Sequencing Project (ESP). A definition of MAF can be found here: (http://www.ncbi.nlm.nih.gov/projects/SNP/docs/rs_attributes.html. Briefly, dbSNP is reporting the minor allele frequency for each rs included in a default global population. Since this is being provided to distinguish common polymorphism from rare variants, the MAF is actually the second most frequent allele value. In other words, if there are 3 alleles, with frequencies of 0.50, 0.49, and 0.01, the MAF will be reported as 0.49. The default global population is 1000Genome phase 1 genotype data from 1094 worldwide individuals, released in the May 2011 dataset.
MS/MS Validation of MiHA Sequences.
The highest scored MS/MS spectra of all candidate MiHAs detected in at least one of the individuals were validated manually, using Xcalibur™ software version 2.2 SP1.48 (Thermo Scientific®). MS/MS spectra of the selected MiHAs were further validated using synthetic MiHA versions synthesized by Genscript. Subsequently, 250-500 fmol of each peptide were injected in the LTQ-Orbitrap ELITE™ or the Q-Exactive™ Plus mass spectrometer using the same parameters as those used to analyze the biological samples.
Determination of the Tissue Distribution of Gene Expression.
Allogeneic T cells can react against a multitude of host MiHAs expressed elsewhere than in hematopoietic/lymphoid organs and induce GVHD. Therefore, to avoid GVHD MiHA expression should not be ubiquitous. Unfortunately, current technical limitations prevent from experimentally assessing MiHA expression in these tissues by mass spectrometry. As an alternative, it was previously shown that MAPs preferentially derive from abundant transcripts (Granados et al. Blood 2012). Thus, the level of expression of transcripts coding for MiHAs could be used as an indicator of MiHAs expression. Publicly available data from Fagerberg et al., Mol Cell Proteomics 2014 13: 397-406 were used, which is part of The Human Project Atlas (THPA) (http://www.proteinatlas.org/tissue, Uhlen et al (2010). Nat Biotechnol. 28(12):1248-50), listing the expression profiles of human genes for 27 tissues. From this data, the expression level of genes coding for the identified MiHAs was obtained. Genes were then classified as “ubiquitous” if expressed in 27 tissues with a “Fragments Per Kilobase of exons per Million mapped reads (FPKM)”>10 or as “not ubiquitous” if not expressed with a FPKM>10 in all 27 tissues. Also, these data were used to calculate the ratio of MiHA genes expression in the bone marrow compared to the skin. Of note, the bone marrow samples used by from Fagerberg et al. (supra) were Ficoll™-separated preparations in which non-hematopoietic components of stroma, adipose cells, bone and vessels, as well as large portions of the fully differentiated erythropoietic and myelopoietic populations had been removed (http://www.proteinatlas.org/humanproteome/bone+marrow). Reads Per Kilobase per Million mapped reads (RPKM) values of MiHA-coding genes in AML samples were obtained from the TCGA Data Portal version 3.1.6. AML data included 128 samples of different subtypes: 12 MO, 36 M1, 29 M2, 12 M3, 23 M4, 14 M5, 2 not classified. Values were converted to Log10(1,000 RPKM+1) for visualization purposes. Mean values were calculated using the 128 AMLs, expect for the Y chromosome-encoded UTY gene, for which only 65 male samples were considered.
Cumulative Number of Identified MiHAs Per Individual.
A custom software tool was used to estimate the cumulative number of HLA-A*02:01 or HLA-B*44:03-associated MiHAs expected for each additional individual studied. Since this number is influenced by the MiHAs present in each individual and by the order in which individuals are analyzed, we exhaustively listed the number of newly identified MiHAs expected for each additional individual studied in all combinations and permutations of groups of studied individuals. Then, we computed the average number of MiHAs for each number of studied individuals. To approximate the cumulative number of MiHAs for up to 20 individuals, a predictive curve was mapped on the data points. The curve was fitted on a function using the curve_fit tool from the “optimize” module of the “scipy” Python library (Jones E, Oliphant E, Peterson P, et al. SciPy: Open Source Scientific Tools for Python, 2001-, http://www.scipy.org/). The following equation was used to represent the cumulative number of identified MiHAs:
Frequency of Therapeutic MiHA Mismatches.
In order to estimate the number of therapeutic MiHA mismatches, a bioinformatic simulation approach was used. For each ns-SNP encoding the 39 optimal MiHAs, the reported alleles were retrieved from the European-American population of the Exome Sequencing Project (ESP) or, if not available, from the European population of “The 1,000 Genomes Project” (http://www.1000genomes.org/). Next, the alleles were categorized from a peptide perspective as ‘dominant’ if the MiHA was detected by MS or known to be immunogenic, or as ‘recessive’ if the MiHA was neither detected by MS nor shown to be immunogenic. Of note, in some loci both alleles were codominant. It was assumed that the presence of a dominant allele always leads to the surface expression of the MiHA. In the case of overlapping MiHAs deriving from the same ns-SNP, the MiHA locus was considered only once. In this simulation, it was also assumed that MiHA-coding SNPs are independent events. In the case of Y chromosome-derived MiHAs (absent in females), a therapeutic mismatch occurred in all male recipient/female donor pairs. Based on the reported minor allele frequencies (MAFs), the allele frequency of the ‘dominant’ or of the ‘recessive’ MiHA was determined in all MiHA-coding loci. Assuming a female/male ratio of 1:1, 1×106 unrelated donor/recipient pairs were randomly generated and virtually genotyped using increasing subsets (1 to 30) of this ranked list of MiHAs. Thus, one population was generated for each MiHA subset. The MAF of each MiHA was used as a probability to generate each individual's maternal and paternal MiHA alleles. For each MiHA subset tested, this procedure resulted in two sets of MiHA alleles (or MiHAs haplotypes) per individual. The number of MiHA mismatches found in each pair was determined and if at least one mismatch was achieved, a therapeutic mismatch was called. The same procedure was used for the related pairs, except that the sampling population corresponded to the progeny of a parental population and was generated according to Mendelian inheritance. This procedure was repeated 1×106 times for both related and unrelated pairs.
Statistical Analyses and Data Visualization.
Unless otherwise stated, analyses and figures were performed using the RStudio™ version 0.98.1091, R version 3.1.2 and Prism™ version 5.0d software. The Wilcoxon rank sum test was used to compare the polymorphic index distribution of exons and exon-exon junctions, or of MiHA-coding genes and that of genes coding for non-polymorphic MAPs. The gplots package in R was used to perform hierarchical clustering and heatmaps of MiHA genes expression in different AML subtypes. Mean expression of MiHA genes among AML subtypes was compared using ANOVA followed by Tukey's multiple-comparison test.
Determination of the Immunogenicity of the Identified MiHAs.
T cells and monocytes were purified from 100-150×106 PBMCs obtained from MiHA-negative individuals using MACS® cell separation columns (Miltenyi Biotec®) or EasySep™ kits (Stemcell Technologies®). Monocyte-derived dendritic cells were generated as previously described (3) and matured with GM-CSF, IL-4, PGE2, TNFα, IL-1β, IL-6 and IFNγ. These dendritic cells were then irradiated at 40 Gy and pulsed with 2 μg/mL of the synthetic MiHA peptide (GLRX3-1S, WDR27-1L, MIIP-2E, or RASSF1-1S) or an irrelevant peptide (Epstein-Barr virus LMP2426-434) that was used a negative control. Pulsed or unpulsed dendritic cells were then co-cultured with previously enriched autologous T cells (5×105/well) in 48-well plates in advanced RPMI medium supplemented with 10% of human serum, 1% of L-Glutamine and 30 ng/mL of IL-21 at a 1:4 (stimulator:effector) ratio. Supplemented media with IL-7 and IL-15 was added after 3, 5 and 7 days of culture and cells were transferred in 12-well plates and 6-well plates at day 5 and 7, respectively. After 10 days of culture, T cells were harvested to determine antigen reactivity with ELISpot for IFNγ and polyfunctional intracellular cytokine staining. Briefly, ELISpot analysis was performed according to the manufacturer's instructions (MABtech®) and intracellular staining was performed after restimulation with 5 μg/mL of peptide in the presence of Brefeldin A for 4 hours. Subsequently, cells were stained for CD3 and CD8 surface markers, and with antibodies directed against the following cytokines for intracellular staining (obtained from BD Biosciences®): IFNγ (Ab 4S.B3), IL-2 (Ab MQ1-17H12), TNFα (Ab MAb11), for intracellular staining. Acquisition was performed with a BD™ LSR II flow cytometer and data were analyzed using Flowlogic™ software (Inivai Technologies®).
A MiHA is essentially a MAP coded by a genomic region containing an ns-SNP.13,21 All human MiHAs discovered to date derive from bi-allelic loci with either two co-dominant alleles or one dominant and one recessive allele.21,26 Indeed, an ns-SNP in a MAP-coding sequence will either hinder MAP generation or generate a variant MAP.11 Hence, at the peptidomic level, each allele can be dominant (generate a MAP) or recessive (a null allele that generates no MAP). All MiHAs reported in this work were detected by MS and are therefore coded by dominant alleles. It was reasoned that two features should dictate which of these MiHAs may represent adequate targets for immunotherapy of HCs. First, the usefulness of a MiHA is determined by the allelic frequency of the MiHA-coding ns-SNP. Indeed, in order to be recognized by allogeneic T cells, a MiHA must be present on host cells and absent in donor cells (otherwise, donor T cells would not recognize the MiHA as non-self). This situation is referred to as a “therapeutic mismatch”. The probability to have a therapeutic mismatch is maximal when the allelic frequency of the target MiHA is 0.5 and decreases as the allele frequency approaches the two extremes of 0 and 1.14 Thus, MiHA having an allele frequency of 0.01 or 0.99 would yield a low frequency of therapeutic mismatch: in the first case, MiHA-positive recipients would be rare whereas in the second case, MiHA-negative donors would be difficult to find. As a rule only variants with a MAF≧0.05 are considered as common and balanced genetic polymorphisms.33 Thus, all MiHAs coded by loci whose least common (minor) allele had a frequency<0.05 were excluded from further analyses. Second, the tissue distribution of a MiHA is relevant to both the efficacy and the innocuity of MiHA targeting. For HC immunotherapy, the target MiHA must be expressed in hematopoietic cells (including HC cells) but should not be ubiquitously expressed by host tissues.
Proteogenomic analyses were performed on B lymphoblastoid cell lines (BLCLs) from 13 individuals expressing HLA-A*02:01 and/or HLA-B*44:03 allotypes. About 55% of European Americans express at least one of these two allotypes. Whole exome and transcriptome sequencing was performed for each cell line in order to identify ns-SNPs and then in silico translated the genomic sequences to create personalized proteomes. Each proteome was subsequently used as a reference to sequence the individual-specific repertoire of MAPs by high-throughput MS.26 A total of 6,773 MiHA candidates generated by ns-SNPs were identified by MS. However, 96.2% of these ns-SNPs were of limited clinical interest because they were rare variants with a MAF<0.05 (
R/QLAPTLSQL
I/SLAPCKLETV
S/ILAPCKLETV
E/QETAIYKGDY
N/DEVLIHSSQY
T/ILLEDGTFKV
I/TLLEDGTFKV
A/SEIEQKIKEY
S/AEIEQKIKEY
R/LLQEELEKL
Q/EEAPESATVIF
Q/REYQVKLQA
R/QEYQVKLQA
L/M/VEADLPRSW
1The residues in bold and separated by “/” indicate the amino acid variation(s) present in the MiHA.
2The genes from which these MiHAs are derived are located on chromosome Y. Accordingly, this MiHa is present in male but absent in female individuals.
3For the MiHAs derived from genes located on chromosome Y, the positions indicated correspond to the position of the first residue of the peptide in the protein, or the position of the first nucleotide encoding the first residue of the peptide in the transcript.
As a proof of principle, the immunogenicity of four novel MiHAs was tested: GLRX3-1S, MIIP-2E, RASSF1-1S and WDR27-1L (
Previous MiHA discovery efforts have largely focused on HLA-A*02:01 and to a lesser extent on HLA-B*44:03.14,24,34. The proteogenomic approach used herein increased the total number of MiHAs presented by HLA-A*02:01 from 21 to 52, and by HLA-B*44:03 from 4 to 67 (
It was assumed that, for HC immunotherapy, optimal MiHAs should be expressed on hematopoietic cells, including the target HC cells, but should ideally not be ubiquitously expressed. Indeed, ubiquitous expression decreases the efficacy of immunotherapy by causing exhaustion of MiHA-specific T cells and entails the risk of toxicity toward normal host epithelial cells (Graft-versus-Host-Disease, GvHD). Since the abundance of a MAP shows a good correlation with the abundance of its source transcript,22,38-40 and RNA-Seq is currently the most accurate method for evaluation of transcript abundance, the expression level of MiHA-coding transcripts was evaluated by RNA-Seq. No RNA-Seq data are available for purified primary epithelial cells from all anatomic sites, but this information is available for entire tissues and organs. Publicly available RNA-Seq data on 27 human tissues from different individuals30 were therefore used to assess the expression profile of genes coding the 119 high-frequency MiHAs presented by the HLA-A*02:01;B*44:03 haplotype (94 reported herein and 25 previously reported) (
R/PTLDKVLEV
S/LLAVAQDLT
To evaluate the relative expression of MiHA-coding genes in hematopoietic vs. epithelial cells, RNA-Seq data obtained from bone marrow vs. skin cells were used. Skin cells are not a pure population of epithelial cells (they contain cells of monocytic and dendritic cell lineage), but are nevertheless highly enriched in epithelial relative to hematopoietic cells. As a criterion for preferential expression in hematopoietic cells, an expression ratio 2 in the bone marrow relative to the skin was used. Out of 119 MiHAs, 39 (32.8%) were non-ubiquitous and overexpressed in hematopoietic cells (
Acute myeloid leukemia (AML) is the most common indication for AHCT according to the Center for International Blood and Marrow Transplant Research (CIBMTR, http://www.cibmtr.org). The expression of genes coding the most promising MiHAs in AML cells was thus analyzed using RNA-Seq data from 128 AML samples available from The Cancer Genome Atlas (TCGA) (
NESNTQKTY
S/AEIEQKIKEY
In the cohort of 13 individuals (ten HLA-A*02:01-positive and seven HLA-B*44:03-positive) used in the present study, 94 novel high-frequency MiHAs were identified. It was calculated that by increasing the number of individuals to 20 for each of these two allotypes, it may be expected to increase the total number of high-frequency MiHAs to a maximum of 125 (
It was next assessed whether the set of 39 optimal MiHAs defined in the present study is sufficient for MiHA-targeted immunotherapy of most patients. One million transplantation cases between related or unrelated HLA*02:01/HLA-B*44:03-positive European-American donor-recipient pairs were randomly simulated, and the number of therapeutic MiHA mismatches found in each case was determined. As shown in
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
The present application claims the benefit of U.S. provisional application Ser. No. 62/113,727 filed on Feb. 9, 2015, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2016/050116 | 2/9/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62113727 | Feb 2015 | US |
