WO2009051555A2 - Peptides modifiés se liant à la classe i du cmh - Google Patents

Peptides modifiés se liant à la classe i du cmh Download PDF

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WO2009051555A2
WO2009051555A2 PCT/SE2008/051164 SE2008051164W WO2009051555A2 WO 2009051555 A2 WO2009051555 A2 WO 2009051555A2 SE 2008051164 W SE2008051164 W SE 2008051164W WO 2009051555 A2 WO2009051555 A2 WO 2009051555A2
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Prior art keywords
peptide
polypeptide
seq
hla
sequence
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PCT/SE2008/051164
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WO2009051555A3 (fr
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Daniel Badia-Martinez
Adnane Achchour
Thorbald Van Hall
Marianne Van Stipdonk
Rienk Offringa
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Akademisch Ziekenhuis Leiden
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A61K39/001192—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2

Definitions

  • the present invention relates to peptides modified to enhance their immunogenicity, particularly binding affinity to Class I major histocompatibility complex (MHC-I) , and methods for modifying peptides to enhance their immunogenicity, particularly MHC-I binding affinity.
  • modified peptides may enhance CD8 + T lymphocyte response useful in the treatment of various pathologies including infections and cancer.
  • MHC-I molecules are plasma membrane proteins expressed by virtually all mammalian cells. They bind peptides derived by intracellular processing of viral, bacterial or endogenous proteins. Their main function is to transport and present these peptides to CD8+ T lymphocytes.
  • the interaction of peptide-MHC protein complexes on the surface of professional antigen presenting cells (APC) with antigen receptors on the surface of T-cells causes T-cell activation and stimulation of an immune response.
  • APC professional antigen presenting cells
  • MHC-I molecules play a crucial role in immune surveillance by selectively binding to intracellular peptide antigens and presenting them at the cell surface to CD8 + T lymphocytes, including cytotoxic T lymphocytes (CTLs) , through their T cell receptor (TCR) .
  • CTLs cytotoxic T lymphocytes
  • TCR T cell receptor
  • tumour-associated antigens recognised by CTLs at the molecular level opens new possibilities for the design of well-defined therapeutic cancer vaccines. Eradication of tumours is often associated with a robust CTL response to TAAs. But since many TAAs are self-proteins or closely related to self-proteins, they tend to be poorly immunogenic. Moreover, many TAA-derived peptides are not strong binders to MHC-I molecules making them poor inducers of CD8 T-cell immunity.
  • the first two domains ( ⁇ l and ⁇ 2 ) of the MHC Class I proteins form an extremely polymorphic peptide-binding groove with closed ends, approximately 30 A long and 12 A wide in the middle.
  • Each ⁇ -helical domain contributes half of the eight-stranded ⁇ -sheet floor of the groove and an ⁇ -helix wall.
  • the most variable residues point into this groove and up from the tops of both helices, conferring unique peptide- and TCR-binding properties to each MHC molecule.
  • the majority of these variable residues are located in the central portion of the cleft, whereas clusters of highly conserved residues that hold on to the peptide termini occur at both ends of the groove.
  • Some of these conserved, primarily aromatic, residues block the ends of the binding groove, while others create a conserved network of hydrogen-bonding ligands and water molecules whereby the termini of the peptides are rigidly fixed.
  • Residues that comprise each region are approximately the same in all the defined MHC-I structures, so that each section is located at the same place in the binding cleft in any MHC Class I molecule. All the structures have two distinct and fairly conserved pockets A and F at each end of the peptide-binding groove. Pockets B through E have distinct sizes and character in different allelic variants of MHC Class I molecules, thereby imposing different sequence constraints and requirements on the peptide that is bound. A consequence of this is that MHC-I binding peptides contain allele-specific sequence motifs, defined by the position and the identity of at least a couple of anchoring residues, one of which is the C-terminus .
  • gplOO is an enzyme involved in pigment synthesis that is expressed by the majority of malignant melanoma cells, as well as by normal melanocytes. gplOO is a member of a family of "self" (i.e., unmutated) , melanoma/melanocyte differentiation antigens that are widely expressed by melanoma cells. Overwijk et al, (J. Exp. Med.
  • TAP-deficient RMA/S cells and a 3-log increase in IFN ⁇ release by recognising T-cells The two peptides are identical in 6 positions of 9, but are different in positions 1, 2 and 3. (Conventionally the residues of an MHC-displayed peptide are numbered sequentially with position 1 being the first residue localised within the MHC-I binding groove.) Peptides that bind to H-2D b commonly use positions 5 and 9 as main anchor positions (with most often an asparagine at position 5 and a hydrophobic residue at position 9) .
  • the present inventors have found that one or more modifications may be introduced in a peptide sequence of a CD8+ T cell epitope of an antigen, wherein the modifications enhance binding affinity of the peptide for MHC-I, and which may moreover generate an enhanced CD8+ T cell immune response.
  • the modifications may be such that the conformation of the modified peptide, when presented, preserves T cell recognition.
  • a polypeptide or peptide comprising the modified peptide sequence may induce a stronger response from CD8 + cytotoxic T lymphocytes (CTLs) towards the polypeptide or peptide comprising the modified peptide sequence as compared with the parent peptide.
  • CTLs cytotoxic T lymphocytes
  • the modified peptide sequence may also indirectly generate a stronger response from other immune cells. Such a response may be directed towards both the modified and the original peptides.
  • the modified peptide sequence may generate an enhanced immune response in an individual, which may be of therapeutic advantage for the treatment of pathologies such as cancer or infection, including use in vaccines.
  • Increased binding affinity of a peptide for MHC-I is additionally advantageous for in vitro applications which exploit or require stability of the peptide-MHC-I interaction, such as the use of MHC tetramers e.g. in fluorescence activated cell sorting (FACS) .
  • FACS fluorescence activated cell sorting
  • the present invention provides a method of producing a polypeptide or peptide, or nucleic acid encoding the polypeptide or peptide, wherein the polypeptide or peptide comprises a modified peptide sequence that has increased binding affinity for an MHC-I molecule and/or generates a higher CD8+ T cell immune response, the method comprising: identifying a peptide sequence ("parent peptide") of a CD8 T cell epitope of an antigen, wherein the peptide sequence is capable of being displayed on an MHC Class I molecule; and producing a polypeptide or peptide comprising a modified peptide sequence of the CD8 + T cell epitope or a nucleic acid molecule encoding said polypeptide or said peptide, wherein the modified peptide sequence has a mutation compared with the parent peptide, the mutation providing a cyclic or pseudo-cyclic amino acid residue at position 3 of the modified peptide sequence, defined such that the residues are sequentially numbered
  • the cyclic amino acid residue may, for example, be a proline or an aromatic residue, such as tyrosine.
  • aromatic cyclic side chains as well as pseudo-cyclic residues at position 3 of the peptide may further stabilize the peptide's interaction with the peptide-binding cleft of the MHC molecule through so-called ring-ring stacking interactions with the side chain of the MHC class I tyrosine residue Y159 (Burley and Petsko, Science, 5 July, pp.23-29, 1985) .
  • This approach of binding improvement should be applicable to all MHC-I independent of specific binding pockets, since the Y159 is conserved among all human, and even mammalian, MHC-I alleles.
  • the invention provides a method of producing a polypeptide or peptide, or nucleic acid molecule encoding the polypeptide or peptide, wherein the polypeptide or peptide comprises a modified peptide sequence that has increased binding affinity for an MHC Class I molecule and/or generates a higher CD8+ T cell immune response, the method comprising: identifying a peptide sequence ("parent peptide") of a CD8 T cell epitope of an antigen, wherein the peptide sequence is capable of being displayed on an MHC Class I molecule and wherein the peptide sequence has a proline, an aromatic (such as tyrosine) , a cyclic or pseudo-cyclic amino acid at position 3, defined such that the residues are sequentially numbered with position 1 being the first residue displayed within the peptide-binding groove of the MHC Class I molecule; and producing a polypeptide or peptide comprising a modified peptide sequence of the CD8 + T cell epitope or an nucleic acid
  • the mutation may comprise providing a proline at position 3 of the peptide, providing a combination of a glycine at position 2 and a proline at position 3 of the peptide, or (for example when the MHC-I molecule comprises HLA-B53) providing a proline at position 2 and a proline at position 3.
  • the mutation may comprise providing arginine or a lysine at position 2 and a proline at position 3.
  • the binding motifs of peptides that bind to some specific MHC-I alleles make use of anchor residues other than at position 2 of the peptide.
  • the peptide may be adapted to the specific allele requirements through the introduction of a proline, an aromatic (such as tyrosine), a cyclic or a pseudo-cyclic residue at position 3 of the presented peptide in accordance with the present invention.
  • the peptide may be modified at other positions so as to provide optimal complementarities between the peptide and the MHC-I allele. Modifications and combinations of modifications may be selected with reference to Table 3.
  • Methods of the invention may further comprise determining binding affinity of the modified peptide for an MHC-I and comparing the binding affinity with the binding affinity of the parent peptide for the MHC-I molecule.
  • the method may comprise determining that the modified peptide has a higher binding affinity than the parent peptide for the MHC-I molecule.
  • methods of the invention may further comprise determining ability of the modified peptide to generate a CD8+ T cell immune response, and comparing the level of CD8+ T cell immune response generated by the modified peptide with the level of CD8+ T cell immune response generated by the parent peptide.
  • the method may comprise determining that the modified peptide generates a higher CD8+ T cell immune response than the parent peptide. Examples of assays for determining binding affinity and for determining CD8+ T cell immune responses are described elsewhere herein .
  • Methods of the invention may be used to determine effects of certain mutations in peptides, in terms of affinity for binding MHC-I and/or for generating CD8+ T cell immune responses.
  • methods of the invention may be used to produce a polypeptide or peptide, or nucleic acid encoding the polypeptide or peptide, wherein the modified polypeptide or peptide comprises a modified peptide sequence, the method comprising identifying a parent peptide and producing a polypeptide or peptide comprising a modified peptide sequence, or an isolated nucleic acid encoding said polypeptide or peptide, wherein the modified peptide sequence has a mutation compared with the parent peptide as discussed elsewhere herein.
  • mutation may provide a proline residue, an aromatic residue (such as tyrosine) , a cyclic or pseudo-cyclic residue at position 3 and/or may provide glycine, or a hydrophobic residue, or any required residue in order to complement the B-pocket, at position 2.
  • the method may comprise determining whether the modified peptide has greater binding affinity for MHC-I and/or generates a higher CD8+ T cell immune response, as compared with the parent peptide.
  • the effect of mutation on binding affinity and/or immune response may vary according to the nature of the mutation, the identity of the MHC-I, and the combination of these two variables.
  • methods of the invention may be used to determine which of the mutations described herein provide the greatest increase in binding affinity and/or immune response, in relation to one or more MHC-I.
  • the invention provides modified polypeptides and/or peptides, and/or nucleic acid molecules encoding modified polypeptides and/or peptides, obtained by methods of the invention.
  • the invention provides D/L polypeptides and/or peptides obtained by methods of the invention.
  • the invention provides polypeptides and/or peptides with reduced peptide-bonds obtained by methods of the invention .
  • the invention provides poly-N-acylated polypeptides and/or peptides obtained by methods of the invention.
  • the invention provides beta amino acid- substituted polypeptides and/or peptides obtained by methods of the invention .
  • the invention provides partially modified retro- inverso pseudo-polypeptides and/or pseudo-peptides obtained by methods of the invention.
  • the invention provides an isolated polypeptide or peptide comprising a mutant peptide sequence which comprises a CD8 + T cell epitope capable of being displayed on an MHC-I, wherein said mutant peptide sequence has the sequence of a naturally occurring CD8 + T cell epitope of an antigen, except that said mutant peptide sequence has a proline, an aromatic residue (such as tyrosine) , cyclic or pseudo-cyclic amino acid residue at position 3 which is not present at position 3 of the sequence of said naturally occurring CD8 + T cell epitope, and optionally has a hydrophobic amino acid residue at specific anchor positions such as position 2 or 9 which is not present at position 2 or 9 of the sequence of said naturally occurring CD8+ T cell epitope, and wherein the position numbering is defined such that the residues are sequentially numbered with position 1 being the first residue displayed within the peptide- binding groove of the MHC Class I molecule.
  • the invention provides an isolated polypeptide or peptide comprising a mutant peptide sequence which comprises a CD8 + T cell epitope capable of being displayed on an MHC-I, wherein said mutant peptide sequence has the sequence of a naturally occurring CD8 + T cell epitope of an antigen, except that said mutant peptide sequence has a proline, an aromatic residue (such as tyrosine) , cyclic or pseudo-cyclic amino acid residue at position 3 which is not present at position 3 of the sequence of said naturally occurring CD8 + T cell epitope, and optionally has a appropriate amino acid residue at specific anchor positions such as an arginine at position 2 of a peptide that binds to HLA-B27 (Lopez de Castro J.
  • the invention provides: an isolated nucleic acid encoding a polypeptide or peptide according to the invention; a vector comprising a nucleic acid according the invention; a host cell comprising a vector according to the invention; and a pharmaceutical composition comprising a polypeptide or peptide of the invention or a nucleic acid of the invention or a vector of the invention and a pharmaceutically acceptable excipient.
  • the invention provides a polypeptide or peptide according to the invention or an isolated nucleic acid encoding a polypeptide or peptide according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention for use in a method of therapy of the human or animal body, particularly for use in generating an immune response in a subject or preventing an infection or a cancer, such as melanoma, in a subject.
  • the invention provides use of a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for generating an immune response in a subject or preventing an infection or a cancer, such as melanoma, in a subject.
  • the invention provides methods of treating an individual comprising administering a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention in order to generate an immune response in the subject or to prevent or treat an infection or a cancer, such as melanoma.
  • the individual may be a human or a non-human animal, e.g. mammal or bird.
  • the invention provides use of a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for generating immunological tolerance in a subject.
  • the subject may have a neurological disease, such as Parkinson's disease or Alzheimer's disease .
  • the invention provides use of a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for preventing or reducing an autoimmune reaction in a subject, wherein the subject has a disease having an autoimmune component.
  • the disease having an autoimmune component may be a disease selected from multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Guillain-barre syndrome and Crohn's disease.
  • the invention provides a complex comprising an MHC-I and a peptide according to the invention.
  • the invention may comprise one or more dimerical, tetramerical or oligomerical complexes comprising an MHC-I molecule and a modified peptide according to the invention.
  • Figure 1 shows the electron density maps of the three peptides, the mouse homologue non-immunogenic mgpl00 25 - 33 (EGS) (A), the intermediate immunogenic human peptide hgplOU 25 - 33 (KVP) (B), and the highly immunogenic heteroclitic peptide modified-gplOO (EGP) (C), all in complex with H-2D b .
  • the annealed omit 2F O -F C electron density map of EGS, KVP and EGP when bound to H-2D b (upper (A) , middle (B) and lower (C) panels, respectively), contoured at 1.0 ⁇ .
  • MHC-peptide complexes are displayed for comparison.
  • the peptides are depicted with their N termini to the left and their C termini to the right.
  • the heavy chain of H-2D b is omitted for simplicity.
  • Figure 2A shows superimposed side views of the peptides mgpl00 2 5-33
  • Figure 2B The side chain of the proline residue at position 3 of the EGP peptide (in grey) has shifted by about 0.8A towards the C-terminal of the peptide binding cleft, allowing for a better interaction with heavy chain residues Y159 and E9.
  • Figure 3A shows the interaction of the low-affinity peptide mgpl00 25 - 33 (EGS) in complex with H-2D b , depicting the interaction between residues 1-5 of the peptide (plE-p5N, indicated in bold in the figure) and residues Yl 1 E9, K66, Q70, Q97, S99 and Y159 from the heavy chain of H-2D b .
  • Distances (in A) between the side chain of the serine residue at position 3 of the peptide (p3S) and surrounding MHC-I residues such as Y159, E9 and S99 are indicated beside the dashed lines.
  • Residues Y45, K66 and Q70, the side chains of which form parts of the binding B-pocket are depicted.
  • Figure 3B shows the interaction of the intermediate-binding affinity peptide hgplOU 25 - 33 (KVP) with H-2D b , identifying potential binding interactions with residues Yl 1 Y22, Y45, K66, Q70 and Y159.
  • Distances (in A) between the side chain of the proline residue at position 3 of the peptide (p3S) and surrounding MHC-I residues such as Y159, E9 and S99 are indicated besides the dashed lines. Note that the side chain of the proline residue p3P in the peptide forms CH- ⁇ and CH-O interactions with the side chain of the heavy chain residues Y159, conserved amongst most of the known MHC Class I molecules .
  • Figure 3C shows the interaction of the very high-binding affinity heteroclitic peptide gplOO (EGP) with H-2D b .
  • the position of the proline residue has shifted by about 0.8A, allowing for a better CH- ⁇ interaction with the side chain of residue Y159.
  • the side chain also interacts through CH-O interactions with the side chain of the MHC Class I glutamate residue E9. Hydrogen bond interactions are depicted as dashed lines.
  • the side chain of proline residue at position 3 of the EGP peptide binds deeper in the cleft of H-2D b , allowing for more extensive van der Waals interactions with MHC-I residues.
  • Figure 4 shows RMA-S binding curves for mgpl00 25 - 33 (EGS) and hgpl00 25 - 33 (KVP), as well as peptides modified at position 3, with H-2D b .
  • the modification of peptide residue p3 from a serine in EGS to a proline in EGP results in a strongly enhanced binding affinity (as indicated by the larger arrow) .
  • Figure 5 shows A) A comparison of the binding affinity of the EGS, EGP and KVP to two other peptides (ElA and MuLV) , known from previous studies to bind very well (ElA) and not at all (MuLV) , respectively, to H-2D b .
  • ElA and MuLV two other peptides
  • the substitution of p3S to p3P in EGP results in the formation of a peptide that binds much better than ElA.
  • the progressive shortening of the side chain of the residue at position 2 of the peptide increases the binding affinity of the modified peptides to H-2D b (compare KVP, KAP and KGP, where KVP is an intermediary binder to H-2D b while KGP shows better binding) .
  • the introduction of a glycine at position 2 of the peptide increases the binding affinity of the modified peptide to H-2D b .
  • the binding affinity increases from a relatively poor binder (EGS) to a very high affinity-binder (EGP) .
  • Figure 6 shows a comprehensive functional analysis of the impact of the different peptide variants KVA, KVP, KAP and KGP on a) the proliferation, b) Interferon-gamma production and c) expression of CD62L.
  • Each column is summarised for the draining lymph nodes, the non draining lymph nodes and the spleen.
  • Introduction of a proline at position 3 of the peptide, conjugated with the introduction of a glycine at position 2 results in an enhanced proliferation, an enhanced production of interferon gamma and an enhanced expression of the maturity marker CD62L (compare KVA and KGP) .
  • Figure 7 shows a comprehensive functional analysis of the impact of the different peptide variants EGS, EVS, EVP and EGP on a) the proliferation, b) Interferon-gamma production and c) expression of CD62L.
  • Each column is summarised for the draining lymph nodes, the non draining lymph nodes and the spleen.
  • Introduction of a proline at position 3 of the peptide, conjugated with the introduction of a glycine at position 2 results in an enhanced proliferation, an enhanced production of interferon gamma and an enhanced expression of the maturity marker CD62L (compare EGS and EGP) .
  • Figure 8 shows a comparative analysis of the capacity of CD8 T cells to bind to tetramers of H-2D b in complex with the non-immunogenic low binding affinity peptide EGS.
  • EGS non-immunogenic low binding affinity peptide
  • Figure 9 shows a comparison of the binding affinity of the Epstein- Barr Virus-associated peptide BMLF-I (259-267, with sequence GLCTLVAML) and modified variants to HLA-A2 on the surface of T2-A2 cells.
  • GLC is the unmodified BMLF-I peptide.
  • P3 is the BMLF-I peptide modified at position 3 to a proline.
  • Y3 is the BMLF-I peptide modified at position 3 to a tyrosine.
  • G2P3 is the BMLF-I peptide modified at positions 2 and 3 to a glycine and a proline, respectively. Both proline and tyrosine modifications at position p3 of the peptide analogues resulted in a better binding when compared to the wild-type peptide.
  • the combined p2Gp3P modification does not bind at all.
  • Figure 10 shows a comparison of the binding affinity of the Epstein Barr virus-associated peptide EBNA-4 (416-424, with sequence IVTDFSVIK) and modified variants to HLA-AIl on the surface of T2-A11 cells, using an MHC class I-specific antibody.
  • IVT is the unmodified EBNA-4 peptide.
  • P3 is the EBNA-4 peptide modified at position 3 to a proline.
  • Y3 is the EBNA-4 peptide modified at position 3 to a tyrosine.
  • G2P3 is the EBNA-4 peptide modified at positions 2 and 3 to a glycine and a proline, respectively. The p3P modification results in a better binding affinity to HLA-AIl when compared to the wild-type peptide.
  • Figure 11a shows the amino acid sequence of human gplOO (SEQ ID NO: 1) .
  • Figure lib shows the amino acid sequence of mouse gplOO (SEQ ID NO: 2) . gi 131981217 I ref
  • KVPRNQWDL human orthologue gplOO-derived peptide KVP
  • EGSRNQWDL mouse epitope EGS
  • AVGALEGSRNQDWLGVPRQL and AVGALKVPRNQDWLGVPRQL longer versions of the EGS or KVP peptides
  • frequencies of EGS-directed, IFN- ⁇ -producing CD8+ T cells were measured by flow cytometry. Means and standard deviation of the groups are plotted for one out of three comparable experiments. Statistical analyses were performed using Graph Pad software .
  • KVP binds with higher affinity to H-2Db than EGS as measured by MHC surface stabilization on RMA-S cells.
  • the ElA and MuLV peptides were used as positive and negative controls, respectively. Binding affinity to H-2Db is expressed as an index of mean fluorescence in presence and absence of peptide.
  • mice were immunized with KVP, KGP, EGS or EGP peptides. After 10 days spleens were harvested, stimulated once with the respective peptides in vitro and tested against the immunizing peptide (vaccine peptide) as well as titrated concentrations of the original peptide EGS. IFN- ⁇ production was measured intracellularly and quantified by flow cytometry. Three mice were included in each group. One representative experiment out of three is displayed.
  • TCR Vb usage of H-2D b /EGS-reactive T-cell pool is diverse and comparable for peptide variants
  • mice C57BL/6 mice were immunized with peptides KVP, KGP or EGP in combination with the adjuvant imiquimod. After 10 days spleens were harvested, stimulated once with respective nanomeric peptides in vitro and then activated with l ⁇ g/ml EGS peptide overnight. Cells were stained for CD8, IFN- ⁇ and with the indicated TCR Vb-specific antibody. IFN- ⁇ -producing CD8 + T-cells were gated and expression of TCR Vb segments was determined. The percentages of Vb-positive cells are depicted as the proportion out of the total pool of reactive cells in each mouse, which was set to 100%. Mean and standard error of the mean (six mice per group) are plotted. The EGS-immunized animals yielded too few reactive cells in order to analyse the Vb usage (less then 5%) .
  • residues in amino acid sequences are sequentially numbered from the N to the C terminus.
  • position 1 in the peptide is defined as the first residue localised within the peptide-binding groove of the MHC Class I molecule. This may be the N-terminal residue of the peptide, with the N-terminus forming hydrogen bond interactions with the hydroxyl groups of heavy chain residues tyrosine 7 and tyrosine 171.
  • the first residue extends out of the peptide-binding cleft of the MHC molecule H-2K b (Achour et al, Immunity, 17, 757-758, 2002; Velloso et al., J. Immunology, 172, 5504-5511, 2004) .
  • the second residue, alanine acts as binding residue number 1
  • the valine residue acts as residue number 2
  • the tyrosine residue acts as peptide residue number 3.
  • residue 1 will always be the first residue displayed within the peptide-binding cleft. If one or more N-terminal residues of the displayed peptide sit outside the cleft, the residue number 1 will be the first position of the peptide that occupies the A-pocket within the cleft.
  • Numbering of MHC-I heavy chains follows the commonly used definition that states that the ⁇ l-domain is usually initiated by a motif of three residues ⁇ GSH' (Glycine-Serine-Histidine) and that the glycine residue is considered as the amino acid residue number 1 in the heavy chain.
  • the present invention provides a mutation of the peptide sequence of an MHC Class I epitope.
  • the present inventors have established the importance of the interaction between the amino acid residue at position 3 of the peptide sequence and the tyrosine residue 159 on the heavy chain, conserved among most known classical and non-classical MHC Class I molecules.
  • the mutation will provide a cyclic residue at position 3, such as proline, tyrosine, phenylalanine, histidine or tryptophan, or a synthetic cyclic amino acid such as a hydroxylated or a methylated histidine (Anderton S., Current Opinion in Immunology, 16, 753-758 (2004) ) .
  • the modification will provide a proline or tyrosine at position 3.
  • Mutation to provide tyrosine at position 3 is preferred in the case of peptides that are restricted to the MHC Class I molecule HLA-A2. Mutation to provide tyrosine at position 3 of such HLA-A2-restricted peptides may increase the binding affinity of the modified peptide for HLA-A2 , when compared to the mutation to provide proline at position 3 of the same peptide. However, it should be noted that mutation to a proline still results in an enhancement of binding affinity when compared to wild-type peptide binding affinity.
  • Mutation to provide proline at position 3 is preferred in the case of peptides that are restricted to the MHC Class I molecule HLA-AIl. Mutation to provide proline at position 3 may increase the binding affinity of the modified peptide for HLA-AIl, when compared to the mutation to provide tyrosine at position 3 of the same peptide ( Figures IOA and lOB) .
  • the modification may provide a "pseudo-cyclic" amino acid (also known as a cyclic-like amino acid) .
  • pseudo-cyclic amino acid means an amino acid which has an unclosed ring structure.
  • pseudo-cyclic amino acids are modified amino acid residues, such as N-alkylated amino acid residues (e.g. N-propyl alanine, N-ethyl glycine, and derivatives) .
  • the unclosed ring structure may be able to interact with the conserved heavy chain residue Y159 of an MHC Class I molecule in order to increase binding affinity of the MHC Class I binding peptide.
  • the pseudo-cyclic residue may have a hydrophobic interaction with the ring of the side chain of the heavy chain residue Y159, increasing the binding affinity of the modified peptide to the MHC-I molecule.
  • the cyclic amino acid may be aromatic.
  • aromatic amino acid residues include tyrosine, tryptophan and phenylalanine.
  • the mutation to provide a cyclic or pseudo-cyclic amino acid at position 3, as defined further herein, may be an amino acid modification, addition, deletion or substitution, and specifically includes the possibility of an amino acid substitution of one cyclic or pseudo-cyclic residue for a different cyclic or pseudo-cyclic residue.
  • the parent peptide may comprise a cyclic or pseudo- cyclic residue at position 3, and the modified peptide sequence may comprise a different cyclic or pseudo-cyclic residue.
  • mutation of a tryptophan residue W to a proline residue P or a tyrosine residue Y at position 3 is an example of a mutation providing a cyclic amino acid residue at position 3.
  • the specified mutations may be the only mutations relative to the parent peptide. In such cases the rest of the peptide sequence is unchanged relative to parent. In other cases, further, unspecified mutations may also be present in the modified peptide as compared with the parent peptide.
  • An example of such additional modifications is the introduction of a glycine residue at position 2 of the peptide. Glycine at position 2 may be of particular advantage in the case of peptides that are restricted to the MHC molecule H-2D b .
  • the coupling of a glycine at position 2 with a cyclic residue, such as a proline or a tyrosine, at position 3 of the peptide will further increase the binding affinity of the peptide to H-2D b .
  • valine, leucine, isoleucine, phenylalanine or any other natural or unnatural hydrophobic residue at position 2 and/or position 9 of the peptide is introduced with a valine, leucine, isoleucine, phenylalanine or any other natural or unnatural hydrophobic residue at position 2 and/or position 9 of the peptide.
  • "Hydrophobic residue” as used herein means an amino acid (naturally occurring or synthetic) that has a non-polar side chain. Examples include: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and cysteine.
  • Preferred hydrophobic residues are: glycine, valine, leucine, isoleucine and phenylalanine.
  • a hydrophobic residue at position 2 and/or 9 may be particularly advantageous in the case of peptides that are restricted to a human MHC Class I molecules such as HLA-A2.
  • a combined hydrophobic residue at position 2 and a lysine residue at position 9 may be particularly advantageous in the case of peptides that are restricted to a human MHC Class I molecules such as HLA-AIl.
  • the introduction of a mutation may be at position 2 and/or position 9 of a peptide that is restricted to HLA-A2 or HLA-AIl, i.e. at both positions 2 and 9, or only at position 2 or only at position 9 of the modified peptide.
  • the provision of a mutation at position 2 of the modified peptide may provide improved complementarity between the modified peptide and the peptide-binding groove within the so-called B-pocket of the MHC Class I molecule.
  • the provision of a mutation at position 9 may provide improved complementarity between the modified peptide and the peptide-binding groove within the so-called B-pocket of the MHC Class I molecule, and the so-called F-pocket, respectively.
  • a number of peptide sequences of CD8 + T cell epitopes of antigens have a cyclic residue at position 3 naturally.
  • the human gpl00 25 - 33 epitope sequence KVPRNQDWL (SEQ ID NO: 7) has proline at position 3.
  • peptide sequence of a CD8 + T cell epitope When a peptide sequence of a CD8 + T cell epitope is identified as having a cyclic residue at position 3 the peptide sequence may be modified at position 2 and/or 9, or in any position of the modified peptide that would result in an increased complementarity between the residue in question and the residues that form any of the pockets with which it interacts. Accordingly, in certain embodiments of the invention
  • the modified peptide sequence may comprise a mutation to provide a glycine residue or a natural or unnatural hydrophobic amino acid residue at position 2 of the modified peptide, or any other fitting residue.
  • the modification is to provide glycine at position 2 in the case of peptides that are restricted to the MHC molecule H-2D b .
  • the modification is to provide a natural or unnatural hydrophobic amino acid residue (such as valine, leucine, isoleucine or phenylalanine) at position 2 and/or position 9 in the case of peptides that are restricted to HLA-A2.
  • the modification is to provide a natural or unnatural hydrophobic amino acid residue (such as valine, leucine, isoleucine or phenylalanine) at position 2 and a lysine residue at position 9 in the case of peptides that are restricted to HLA-AIl.
  • a natural or unnatural hydrophobic amino acid residue such as valine, leucine, isoleucine or phenylalanine
  • a parent peptide having a cyclic or pseudo-cyclic amino acid residue at position 3 is to modify the amino acid at position 3 of the parent peptide to provide a different cyclic or pseudo-cyclic amino acid residue at position 3.
  • the mutation may be to provide a tyrosine at position 3. It has been found that mutation to provide glycine at position 2 of the modified peptide when a proline, a cyclic or cyclic-like residue is present at or is provided at position 3 dramatically increases the binding affinity of the modified peptide for the MHC Class I molecule H-2D b .
  • the preferred mutation at position 2, or any main or secondary anchor position may differ depending on the intended MHC Class I molecule to be bound by the modified peptide.
  • mutation to provide leucine, isoleucine, phenylalanine or valine at position 2 when a cyclic or cyclic-like residue is present at position 3 may increase the binding affinity of the modified peptide for human MHC Class I molecules such as HLA-A2 or HLA-AIl.
  • any mutation described herein may be combined with any other mutation described herein .
  • the parent peptide sequence may be a CD8+ T cell epitope from any antigen of any species that is capable of being displayed, even with an extremely low binding affinity, on an MHC Class I molecule which may be from any of a broad array of species.
  • the antigen may be associated with infection or with a type of cancer or other disease, or associated with an allergic response. In all cases, the antigen has an epitope that can bind, even with an extremely low binding affinity, to an MHC Class I molecule. There may, of course, be more than one such epitope present in the antigen.
  • the peptide sequence of any such epitope or epitopes may be modified in accordance with the above methods in order to enhance immunogenicity .
  • the antigen may be a synthetic peptide or polypeptide having an epitope that can bind to an MHC Class I molecule.
  • Immune Epitope Database IEDB sponsored by NIH, NIAID and HHS: http : / /www . immuneepitope . org/home . do
  • Table 1 provides a non-exhaustive list of tumour-associated antigens which may comprise parent peptide sequences for modification in accordance with the present invention. Polypeptides or peptides comprising modified peptide sequences of these antigens are useful for treatment of the cancers with which the antigens are associated.
  • AAA61244 Britchard, J. Exp. Med., 1993 Tyrosinase related protein-1 AAC154 I Wang, J. Exp. Med., 1995
  • Tyrosinase related protein-2 CAB93531, CAB93530 Wang,
  • MAGE, MAGE family testis and tumor-specific protein, AAK00357 associated with breast, lung, bladder, oesophagus, prostate, colorectal, head and neck cancers.
  • ESO-I Cancer/testis antigen IB (L antigen family member 2) (LAGE-2 protein) , (Autoimmunogenic cancer/testis antigen NY-ESO-I) , P78358; associated to Breast, lung, bladder, oesophagus, prostate head and neck cancers.
  • EBNA-3, -4 and -6 EBNA-3, -4 and -6; EBNA-3, Epstein-Barr nuclear antigen 3 (EBV nuclear antigen 3) (EBNA-3) (EBNA- 3A), P12977; EBNA-4, Epstein-Barr nuclear antigen 4 (CAA373981) , EBNA-6, Epstein-Barr nuclear antigen 6 (P03204), BMLF-I, (ABU55428); associated to nasopharyngeal carcinoma, Burkitt ' s and Hodgkin's lymphoma.
  • EBV nuclear antigen 3 Epstein-Barr nuclear antigen 3
  • EBNA-3A Epstein-Barr nuclear antigen 4
  • EBNA-6 Epstein-Barr nuclear antigen 6
  • BMLF-I BMLF-I
  • HBV surface antigen HBV surface proteins [Hepatitis B virus], CAA48355, HBV core antigen [Hepatitis B virus], CAA59535 and polymerase HBV polymerase [Hepatitis B virus], CAA48354; associated to Hepatoma.
  • HER-2/neu Receptor tyrosine-protein kinase erbB-2 precursor (pl85erbB2) (C-erbB-2) , (NEU proto-oncogene) (Tyrosine kinase-type cell surface receptor HER2), (MLN 19) (CD340 antigen), P04626; associated to breast, ovary, pancreas cancers.
  • Ras muscle RAS oncogene homolog [Homo sapiens], NP 036351; p53, tumor protein p53 [Homo sapiens], NP 000537; associated to many kinds of cancers .
  • MUC-I Homo sapiens
  • CAA56734 associated to breast, pancreas, colon, ovary, lung cancers.
  • SART-I squamous cell carcinoma antigen recognized by T cells 1 [Homo sapiens], NP 005137; associated to squamous head, neck, lung cancers.
  • PRAME preferentially expressed antigen in melanoma [Homo sapiens] NP 996839 and RAGE-I, preferentially expressed antigen in melanoma [Homo sapiens], Q9UQ07; associated with kidney cancer.
  • Telomerase Telomerase protein component 1 (Telomerase-associated protein 1) , (Telomerase protein 1) (p240) (p80 telomerase homolog) , Q99973; associated with many kinds of cancer
  • pathogen-related antigens having MHC-I epitopes are known.
  • Such pathogen-related antigens may comprise parent peptide sequences for modification in accordance with the present invention. Examples of such pathogen-related antigen MHC-I epitopes are shown in Table 9 of patent application GB0720118.9.
  • antigens include the human gplOO antigen having the amino acid sequence of SEQ ID NO: 1 ( Figure lla) and the mouse gplOO antigen having the amino acid sequence of SEQ ID NO: 2 ( Figure lib) .
  • Sequences for hgplOO and mgplOO are also available under database Accession numbers gi
  • a parent peptide sequence of a CD8+ T cell epitope of an antigen may be identified by any suitable method. For example, electronic sequence information may be searched for known antigen sequences, published sequences may be evaluated to identify candidate MHC Class I epitopes, e.g. using an algorithm for the prediction of an MHC Class I epitope. Such algorithms are described further below.
  • human gpl00 2 5-3 3 KVPRNQDWL (SEQ ID NO: 7) and gpl ⁇ 2 o 9 -2i7 : ITDQVPFSV (SEQ ID NO: 23) .
  • MHC Class I epitopes Many different predictive programs are available for the identification of potential MHC Class I epitopes in any given polypeptide. Any suitable method may be used to identify within a given polypeptide or peptide sequence an epitope that is capable of being displayed on an MHC Class I molecule. Examples of epitope- identification programs can be found at the websites of the following organizations and facilities:
  • RANKPEP Reche PA, Glutting JP, Reinherz EL. (2002) 'Prediction of MHC Class I binding peptides using profile motifs' .
  • MHC Class I-restricted peptides are described at the website http://www.immuneepitope.org/home.do. See also Greenbaum et al . Journal of Molecular Recognition 2007 Mar-Apr; 20 (2) : 75-82 , which describes algorithms for epitope identification.
  • Identifying a peptide sequence of a CD8+ T cell epitope may comprise selecting the peptide sequence for modification, and need not comprise predicting a peptide sequence of an epitope, since many peptide sequences of CD8+ T cell epitopes have already been identified, and many examples can be found in the published literature .
  • Table 2 provides a non-exhaustive list of MHC-I epitopes from tumour- associated antigens (TAAs) which may be employed as a parent peptide in accordance with the present invention.
  • TAAs tumour- associated antigens
  • Polypeptides or peptides comprising modified sequences of these parent peptides are useful for treatment of the cancers with which the TAAs are associated.
  • MHC-I epitopes from various pathogen sources are known in the art and a non-exhaustive list is compiled i.a. in Table 9 of patent application GB0720118.9, filed on 15 October 2007.
  • Polypeptides or peptides comprising modified sequences of the parent peptides of Table 9 of GB0720118.9 may be useful for treatment of pathogen- related disease, especially disease associated with the pathogen from which the parent peptide is associated.
  • DAM- 6, -10 (MAGE-Bl, -B2) HLA-A2 FLWGPRAYA (SEQ ID NO: 27)
  • HLA-A3 SLFRAVITK (SEQ ID NO: 32)
  • HLA-A28 EVYDGREHSA (SEQ ID NO: 34)
  • HLA-Cw2 SAFPTTINF SEQ ID NO: 37
  • HLA-Cw3 SAYGEPRKL (SEQ ID NO: 38)
  • HLA-A2 YLQLVFGIEV (SEQ ID NO: 41)
  • HLA-B37 REPVTKAEML SEQ ID NO: 43
  • MAGE-A3 HLA-Al EADPIGHLY SEQ ID NO: 44
  • HLA-A2 FLWGPRALV (SEQ ID NO: 45)
  • HLA-A24 IMPKAGLLI SEQ ID NO: 47
  • HLA-B44 MEVDPIGHLY SEQ ID NO: 48
  • HLA-B*3501 EVDPIGHLY SEQ ID NO: 51
  • MAGE-A4 HLA-A2 GVYDGREHTV SEQ ID NO: 52
  • MAGE-A6 HLA-A34 MVKISGGPR SEQ ID NO: 53
  • HLA-A2 SLLMWITQC (SEQ ID NO: 60)
  • HLA-A2 QLSLLMWIT (SEQ ID NO: 61)
  • HLA-B*3501 MPFATPMEA (SEQ ID NO: 62)
  • CEA HLA-A2 YLSGANLNL (SEQ ID NO: 66)
  • HLA-A3 HLFGYSWYK (SEQ ID NO: 67)
  • Ep-CAM HLA-A2 GLKAGVIAV (SEQ ID NO: 68)
  • GpIOO HLA-A2 KTWGQYWQV SEQ ID NO: 69
  • HLA-A2 AMLGTHTMEV (SEQ ID NO: 70)
  • HLA-A2 MLGTHTMEV (SEQ ID NO: 71)
  • HLA-A2 SLADTNSLAV (SEQ ID NO: 72)
  • HLA-A2 ITDQVPFSV (SEQ ID NO: 73)
  • HLA-A2 LLDGTATLRL (SEQ ID NO: 74)
  • HLA-A2 YLEPGPVTA (SEQ ID NO: 75)
  • HLA-A2 VLYRYGSFSV (SEQ ID NO: 76)
  • HLA-A2 RLMKQDFSV (SEQ ID NO: 77)
  • HLA-A2 RLPRIFCSC SEQ ID NO: 78
  • HLA-A3 LIYRRRLMK (SEQ ID NO: 79)
  • HLA-A3 ALNFPGSQK (SEQ ID NO: 80)
  • HLA-A3 SLIYRRRLMK (SEQ ID NO: 81)
  • HLA-A3 ALLAVGATK (SEQ ID NO: 82)
  • HLA-A* 6801 HTMEVTVYHR (SEQ ID NO: 84)
  • HLA-Cw8 SNDGPTLI (SEQ ID NO: 86)
  • KLLMVLMLA (SEQ ID NO: 88)
  • TTNAIDELK (SEQ ID NO: 89)
  • HLA-A2 EAAGIGILTV (SEQ ID NO: 92)
  • HLA-B45 AEEAAGIGIL (SEQ ID NO: 94)
  • HLA-B45 AEEAAGIGILT (SEQ ID NO: 95)
  • HLA-A2 FLALIICNA (SEQ ID NO: 97)
  • HLA-A2 FLTPKKLQCV SEQ ID NO: 101
  • HLA-A2 VISNDVCAQV SEQ ID NO: 102
  • TRP-I (or gp751 HLA-A31 MSLQRQFLR (SEQ ID NO: 103) TRP-2 HLA-A2 SVYDFFVWL (SEQ ID NO: 104)
  • HLA-A2 TLDSQVMSL (SEQ ID NO: 105)
  • Tyrosinase HLA-Al KCDICTDEY (SEQ ID NO: 109)
  • HLA-A2 YMDGTMSQV (SEQ ID NO: 111)
  • HLA-A2 MLLAVLYCL SEQ ID NO: 112
  • HLA-A24 AFLPWHRLF (SEQ ID NO: 113)
  • HLA-B44 SEIWRDIDF (SEQ ID NO: 114)
  • HLA-B*3501 TPRLPSSADVEF (SEQ ID NO: 115) ⁇ -Actinin-4 HLA-A2 FIASNGVKLV (SEQ ID NO: 116) ⁇ -Catenin HLA-A24 SYLDSGIHF (SEQ ID NO: 117)
  • CDK-4 HLA-A2 ACDPHSGHFV (SEQ ID NO: 119)
  • ELF2 HLA-A68 ETVSEQSNV (SEQ ID NO: 120)
  • HSP70-2 M HLA-A2 SLFEGIDIY (SEQ ID NO: 121)
  • HLA-Cw6 FRSGLDSYV (SEQ ID NO: 127)
  • N-RAS HLA-Al ILDTAGREEY (SEQ ID NO: 133)
  • OGT HLA-A2 SLYKFSPFPL (SEQ ID NO: 134)
  • TGFaRII HLA-A2 RLSSCVPVA SEQ ID NO: 1355
  • TRP-2-6b HLA-A2 ATTNILEHY (SEQ ID NO: 137)
  • Adipophilin HLA-A2 SVASTITGV (SEQ ID NO: 138)
  • HLA-A24 DFMIQGGDF (SEQ ID NO: 145)
  • EphA2 HLA-A* 0201 IMNDMPIYM (SEQ ID NO: 146)
  • FGF-5 HLA-A3 NTYASPRFKb (SEQ ID NO: 148) G250 HLA-A2 HLSTAFARV (SEQ ID NO: 149) GnT-V HLA-A2 VLPDVFIRC (SEQ ID NO: 150) HER-2/neu HLA-A2 KIFGSLAFL (SEQ ID NO: 151)
  • HLA-A2 RLLQETELV (SEQ ID NO: 153)
  • HLA-A2 WLGWFGI SEQ ID NO: 1544
  • HLA-A2 ILHNGAYSL (SEQ ID NO: 155)
  • HLA-A2 YMIMVKCWMI (SEQ ID NO: 156)
  • HST-2 FGF-6 HLA-A31 YSWMDISCWI (SEQ ID NO: 159) hTERT HLA-A2 ILAKFLHWL (SEQ ID NO: 160)
  • HLA-A2 RLVDDFLLV SEQ ID NO: 162
  • HLA-A3 KLFGVLRLK (SEQ ID NO: 163)
  • iCE HLA-B7 SPRWWPTCL (SEQ ID NO: 164)
  • HLA-A2 RLASFYDWPL (SEQ ID NO: 166)
  • M-CSF HLA-B*3501 LPAVVGLSPGEQEY SEQ ID NO: 167; MUCl HLA-AIl STAPPAHGV (SEQ ID NO: 168)
  • HLA-A2 SLYSFPEPEA SEQ ID NO: 1744
  • HLA-A2 ALYVDSLFFL (SEQ ID NO: 175)
  • PSMA HLA-Al HSTNGVTRIY SEQ ID NO: 177)
  • HLA-A24 LYSDPADYF (SEQ ID NO: 178)
  • RAGE HLA-B7 SPSSNRIRNT SEQ ID NO: 183
  • RUl HLA-B51 VPYGSFKHV SEQ ID NO: 184
  • RU2 HLA-B7 LPRWPPPQL SEQ ID NO: 185)
  • SART-I HLA-A24 EYRGFTQDF SEQ ID NO: 186)
  • HLA-A* 2601 KGSGKMKTE SART -2 HLA-A24 DYSARWNEI (SEQ ID NO: 188) HLA-A24 AYDFLYNYL (SEQ ID NO: 189) HLA-A24 SYTRLFLIL (SEQ ID NO: 190)
  • HLA-A2 SAWISKPPGV Survivin HLA-A2 ELTLGEFLKL (SEQ ID NO: 196) HLA-A2 TLPPAWQPFL (SEQ ID NO: 197)
  • TRG HLA-B52 YQLCLTNIF (SEQ ID NO: 199)
  • HLA-A2 RMFPNAPYL SEQ ID NO: 200
  • HLA-A24 CMTWNQMNL SEQ ID NO: 201
  • HLA-A24 RWPSCQKKF SEQ ID NO: 202
  • HLA-A2 MLTNSCVKL (SEQ ID NO: 208)
  • bcr-abl p210 (b3a2)
  • HLA-A2 SSKALQRPV (SEQ ID NO: 209)
  • HLA-A3 ATGFKQSSK (SEQ ID NO: 210)
  • HLA-A3, HLA-AIl HSATGFKQSSK (SEQ ID NO: 212;
  • ETV6/AML HLA-A2 RIAECILGM (SEQ ID NO: 215)
  • SLAMLDLLHV SEQ ID NO: 2136
  • HLA-Bl 4 SSRTRRETQL (SEQ ID NO: 224)
  • HLA-B27 GRWTGRCMSC (SEQ ID NO: 225)
  • HLA-AIl YVNVNMGLK (SEQ ID NO: 230)
  • HLA-A24 EYLVSFGVW (SEQ ID NO: 231)
  • HLA-A24 KYTSFPWLL (SEQ ID NO: 232)
  • HLA-A31 STLPETTVVRR (SEQ ID NO: 233)
  • HLA-A2 WLSLLVPFV (SEQ ID NO: 235)
  • HLA-A2 FLLTRILTI SEQ ID NO: 2336
  • HLA-A3 RLRAEAQVK (SEQ ID NO: 239)
  • HLA-A24 RYSIFFDY (SEQ ID NO: 240)
  • HLA-B7 VPAPAGPIV (SEQ ID NO: 243) HLA-B7 RPPIFIRRL (SEQ ID NO: 244)
  • HLA-B8 YIKSFVSDA (SEQ ID NO: 248)
  • HLA-Bl 8 DEVEFLGHY (SEQ ID NO: 256)
  • MHC-I-binding epitopes have a peptide sequence of 8 to 10 amino acids, with epitopes of 9 amino acids in length being common. However, it has been found that sequence lengths outside this range can also bind to an MHC Class I molecule. For example, Glithero et al., J. Biol. Chem., 2005, Vol. 281, No. 18, pp. 12699-12704 describes binding between a pentapeptide NYPAL and H-2D b .
  • the parent peptide sequence is from 3 to 25 amino acids in length, such as 5 to 15 amino acids in length.
  • the parent peptide is from 8 to 10 amino acids in length.
  • MHC-I MHC Class I molecule
  • the MHC-I may be human.
  • the MHC-I is one having the conserved tyrosine 159 residue.
  • Preferred MHC-I include human MHC-I such as HLA-A2 and HLA- AIl as well as mouse MHC-I such as H-2D b .
  • MHC molecules in different species are listed for example in the following internet sites mentioned above: http : //www. syfpeithi . de/ http : //www. immuneepitope . org/home . do
  • the MHC Class I molecule may be other than H-2D d .
  • the MHC-I may be a classical MHC-I such as H-2D b , HLA-A2 and HLA-AIl.
  • the MHC Class I molecule may be a non-classical MHC-I such as HLA-G, HLA-F, HLA-E, or any novel MHC Class I-like molecule discovered in humans, as well as all MHC Class I-like molecules in vertebrates and/or those produced by viruses, bacteria and any other pathogens .
  • the polypeptide or peptide of the invention may bind to an MHC Class I molecule other than H-2D d .
  • the polypeptide or peptide of the invention may have greater binding affinity for an MHC Class I molecule other than H-2D d than the binding affinity it has for H-2D d .
  • the polypeptide or peptide of the invention has at least 2-fold, such as 5-fold or 10-fold, greater binding affinity for an MHC-I other than H-2D d than the binding affinity it has for H-2D d .
  • Binding affinity of a peptide for an MHC-I can be defined using a competition-based cellular binding assay (van der Burg et al, Human Immunology, 1995; Kessler et al, J. Exp. Med. 2001) .
  • High affinity binding peptides were identified as those with an IC 50 ⁇ 6 ⁇ M, peptides that bound with intermediate affinity corresponded to 6 ⁇ M ⁇ IC 50 ⁇ 15 ⁇ M, peptides that displayed a low binding affinity corresponded to 15 ⁇ M ⁇ IC 50 ⁇ 100 ⁇ M) , and finally peptides with undetectable binding capacity corresponded to an IC 50 > 100 ⁇ M.
  • peptide-MHC stability can be assessed by measuring the dissociation rate of high affinity binding peptides complexed with the MHC Class I molecule at 37°C (van der Burg et al, J. Immunology, 1996) .
  • a modified peptide may be considered as having increased binding affinity for an MHC-I as compared with the parent peptide if the modified peptide exhibits a statistically significant increase in binding affinity as measured in a binding assay as described herein.
  • a modified peptide will have binding affinity for an MHC-I which is at least 10% greater, more preferably at least 50% greater, more preferably at least 100% greater, more preferably at least 5-fold greater, most preferably at least 10-fold greater than the binding affinity of the parent peptide for the same MHC-I.
  • the A-, B-, C-, D-, E- and F-pockets are corresponding to the initial description in the pioneering publication of Prof. Pamela Bjorkman (Bjorkman et al, Nature, 1987 (a) ; Bjorkman et al, Nature, 1987 (b) and Saper et al J. MoI. Biol., Bjorkman et al, J. Immunology, 2005) .
  • structural knowledge acquired since 1987 demonstrated that in some cases, the pockets and in particular pocket B is formed by other residues on the heavy chain of the MHC Class I molecules.
  • Each MHC allele has a distinct peptide specificity that is a reflection of the chemical composition of the residues that comprise the antigen-binding site.
  • This peptide specificity may be summarized by a peptide motif that demonstrates the restrictions and preferences of the individual pockets that comprise the antigen-binding site.
  • Residues that protrude into specificity pockets are termed anchors, and these anchors are characterized by residues or residue classes that are conserved at equivalent positions of the peptide.
  • the term anchor originated from the strong belief that these positions conferred significant binding energy. These anchors may be dominant, whereby an inherent inflexibility in the type of residue chelated suggests highly specific and efficient binding, or secondary or promiscuous whereby a large number of residue types may be accommodated, but some preferences are evident.
  • Each allelic form of a class I molecule preferentially binds peptides that conform to a particular binding sequence motif, often defined by the second (P2) and the last residues (P ⁇ ) , usually P8, P9 or PlO in the human and by the fifth (P5) and the last one (P ⁇ ) in the mouse.
  • Footnote A: alanine, F: phenylalanine, H. histidine, I: isoleucine, K: lysine, L: leucine, M: methionine, N: asparagine, P: proline, R: arginine, S: serine, T: threonine, V: valine, W. tryptophane, Y: tyrosine.
  • any suitable method of producing a polypeptide or peptide comprising the modified peptide sequence or an isolated nucleic acid molecule encoding the polypeptide or peptide may be used in the present invention.
  • the polypeptide or peptide or isolated nucleic acid molecule may be synthesised de novo using knowledge of the sequence of the parent peptide or may be synthesised by mutating the parent peptide sequence e.g. using site-directed mutagenesis. Therefore, a polypeptide or peptide comprising modified peptide sequence may be provided directly with the mutation (s) or indirectly by first producing the parent peptide sequence and mutating it.
  • peptides it may be convenient to synthesise the entire peptide comprising the modified peptide sequence using standard Fmoc chemistry.
  • Another convenient way of providing a longer peptide or a polypeptide is to express nucleic acid encoding it, by use of the nucleic acid in an expression system.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds., John Wiley & Sons, 1992.
  • Nucleic acid encoding a polypeptide or peptide comprising the modified peptide sequence may be produced synthetically making use of the known sequence of the parent peptide.
  • the nucleic acid encoding the parent peptide may be cloned and subjected to site-directed mutagenesis so as to provide an altered nucleic acid sequence encoding the modified peptide sequence.
  • the polypeptide provided comprises the full length sequence of the antigen with the modified peptide.
  • the method may include providing a full length mouse gplOO polypeptide sequence wherein the serine at position 3 of the gpl00 25 - 33 epitope (position 27 of the mouse gplOO sequence) is mutated to proline.
  • the polypeptide provided by the method may be designated mgplOO S27P.
  • the method may provide a fragment of the full length sequence of the antigen wherein the fragment comprises the modified peptide sequence.
  • the fragment may be at least 100 amino acids in length.
  • the modified peptide sequence may be at any suitable position within the polypeptide sequence.
  • the modified peptide sequence may be N-terminal or C-terminal or may lie at any position therebetween.
  • the position of the modified peptide sequence will be chosen such that, upon antigen processing of the polypeptide, the modified peptide sequence is displayed on an MHC Class I molecule.
  • the method may provide a peptide of up to 25 amino acids in length wherein the peptide comprises the modified peptide sequence.
  • the method may provide a peptide having one or more amino acids N-terminal and/or C-terminal of the modified peptide sequence.
  • the method may provide a peptide consisting of the modified peptide sequence.
  • a polypeptide or peptide comprising a modified peptide sequence is said to "correspond to" the full length sequence of the antigen or a fragment of the antigen even though the polypeptide or peptide will not have the exact same sequence as the antigen or a fragment of the antigen; the polypeptide or peptide will have at least one modification in accordance with the invention.
  • the only difference between the sequence of the polypeptide or peptide comprising the modified peptide sequence and the antigen or a fragment of the antigen is one or more modifications in accordance with the invention.
  • polypeptide or peptide may comprise a plurality of MHC Class I epitopes.
  • the plurality may include multiple instances of the same modified epitope and/or a number of differing epitopes.
  • polypeptide may comprise an "epitope string" comprising multiple repeats of the modified peptide sequence.
  • the method may provide an isolated nucleic acid encoding the polypeptide (s) or peptide (s) comprising the modified peptide sequence as described herein.
  • an isolated nucleic acid may be in the form of a vector in order to produce the polypeptide or peptide comprising the modified peptide sequence in a host cell and/or in order to deliver the polypeptide (s) or peptide (s) to an organism, including a human. Therefore, the isolated nucleic acid provided by the method may be useful for delivering the polypeptide (s) or peptide (s) comprising the modified peptide sequence to a patient.
  • a vector will comprise a nucleic acid encoding the polypeptide (s) or peptide (s) comprising the modified peptide sequence as described herein operably linked to one or more regulatory sequences.
  • the polypeptide or peptide comprising the modified peptide sequence may elicit an increased response from CD8 + CTLs as compared with the parent peptide.
  • the increased CTL response may include increased CTL proliferation and/or IFN ⁇ production and/or expression of maturity markers such as CD62L.
  • Such CTL responses may be assessed using assays described in further detail below. It has been found that mutation to provide a cyclic or pseudo-cyclic amino acid at position 3 of the modified peptide increases binding affinity of the modified peptide for an MHC Class I molecule while preserving T cell recognition.
  • the conformation of the modified peptide when presented on an MHC Class I molecule may be essentially unchanged as compared with the conformation of the parent peptide when presented on the MHC Class I molecule, such that T cell recognition is preserved.
  • the polypeptide or peptide comprising the modified peptide sequence may be capable of acting as a T helper peptide-epitope .
  • T lymphocytes like that of other cells, may be measured in vitro by determining the amount of 3H-labelled thymidine incorporated into the DNA of cultured cells. Thymidine incorporation provides a quantitative measure of the rate of DNA synthesis, which is usually directly proportional to the rate of cell division.
  • the production levels of interferon gamma reflect the levels of activation of CD8+ T cells.
  • CD8+ T cell maturity may be assessed by measuring the expression of maturity markers such as CD62L.
  • an increased CD8+ T cell response may be detected by determining the level of (i) IFN- ⁇ (ii) 3H-labelled thymidine incorporated into DNA and/or (iii) expression of maturity markers, in cultured CD8+ T cells contacted with an antigen presenting cell displaying the modified peptide as compared with cultured CD8+ T cells contacted with an antigen presenting cell displaying the parent peptide, and detecting a higher level of (i) , (ii) and/or (iii) for T cells stimulated with the modified peptide as compared with T cells stimulated with the parent peptide.
  • a modified peptide may be considered as causing an enhanced CTL response as compared with the parent peptide if the modified peptide exhibits a statistically significant increase in CTL proliferation, IFN-gamma production and/or expression of maturity markers such as CD62L, as measured using assay methods described herein.
  • a modified peptide will induce a CTL response which is at least 10% greater, more preferably at least 50% greater, yet more preferably at least 100% greater, yet more preferably at least 5-fold greater, most preferably at least 10-fold greater than the corresponding CTL response of the parent peptide.
  • the present invention provides a method of treating a subject which comprises administering an effective amount of a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention.
  • Conditions which may be treated in accordance with the invention include tumours (preferably, cancerous tumours such as melanoma) and infections and conditions alleviated by generation of an immune response.
  • the parent peptide sequence may be a CD8+ T cell epitope from a tumour-associated antigen which is found on the tumour to be treated.
  • the parent peptide sequence may be from gplOO.
  • the present invention provides a method of treating a subject which comprises administering an effective amount of a polypeptide or peptide according to the invention or an isolated nucleic acid according to the invention or a vector comprising a nucleic acid according the invention or a pharmaceutical composition according to the invention.
  • Conditions which may be treated in accordance with the invention include induction of tolerance, in order to stop, slow down and/or impair the progression of autoimmune reactions.
  • the parent peptide sequence may be a CD8+ T cell epitope from a self, a virus, a bacteria or any other pathogen-associated antigen which is correlated to the treated disease.
  • the parent peptide sequence may be from e.g. myelin basic protein (MBP) , proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) .
  • MBP myelin basic protein
  • PGP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • agent or pharmaceutical composition comprising the agent may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or topically (i.e., at the site of desired action) .
  • parenteral administration e.g. injection
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc .
  • Adjuvant s such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc .
  • a polypeptide or peptide of the invention or an isolated nucleic acid of the invention is provided in the form of a pharmaceutical composition comprising an adjuvant.
  • the composition may comprise the polypeptide (s) or peptide (s) comprising the modified peptide (s) sequence (s) and CpG oligodeoxynucleotide as described in Example 3.
  • Non-exclusive examples of adjuvants as well as listings of adjuvants that may be suitable for use with the peptides of the invention can be found the following references: Cox and Coulter, ⁇ Adjuvants - a classification and review of their modes of action', Vaccine, 1997; Singh and O'Hagan, 'Advances in vaccine adjuvants', Nature Biotechnology, 1999.
  • Alum aluminium-based mineral salts aluminium hydroxide, aluminium phosphate, calcium phosphate.
  • Cytokines e.g. IL-2, IL- 12 and GM-CSF
  • Saponins e.g. QS21
  • CpG oligos Lipopolysaccharide (LPS)
  • MPL Monophosphoryl Lipid A
  • Polyphosphazenes e.g. IL-2, IL- 12 and GM-CSF
  • Emulsions e.g. Freund's, SAF, MF59
  • Liposomes e.g. Virosomes, Iscoms Cochleates.
  • PLG polylactide co-glycolide microparticles PLG polylactide co-glycolide microparticles, Poloxamer particles, Virus-like particles.
  • Mucosal adjuvants Heat-labile enterotoxin (LT) , Cholera toxin (CT) , Mutant toxins (e.g. LTK63 and LTR72) , microparticles, polymerised liposomes.
  • LT Heat-labile enterotoxin
  • CT Cholera toxin
  • Mutant toxins e.g. LTK63 and LTR72
  • Non-particulate adjuvants Muramyl dipeptide (MDP) and derivatives. Other adjuvants
  • Non-ionic block copolymers e.g. polyoxypropylene/polyoxyethylene .
  • a soluble, multimeric complex of MHC Class I molecules and peptides according to the invention may be used in identification and characterisation of antigen-specific T cells.
  • the tetramers may bind peptide-specific cytotoxic T cells in vitro. The use of tetramer assays to assess immunisation responses is described in further detail in Example 5 below.
  • Example 1 Crystal structures of MHC-I molecule in complex with gplOO peptides
  • the present inventors initiated a structural study involving the determination of the crystal structures of an MHC-I molecule from the mouse, H-2D b , in complex with two 9-mer variants of a peptide derived from the melanoma-associated protein gplOO.
  • the reasons underlying the differences in binding affinity and immunogenicity of mouse gpl00 25 - 33 (mgplOO corresponding to the sequence EGSRNQDWL (SEQ ID NO: 3) and human gpl00 25 - 33 (hgplOO corresponding to the sequence KVPRNQDWL, SEQ ID NO: 7) were studied. Both main anchor positions at positions 5 (asparagine) and 9 (leucine) are conserved between the two peptides.
  • H-2D b /peptide MHC complexes Both the H-2D b heavy chain and the murine ⁇ 2 m were expressed in Escherichia coli.
  • the H-2D b /KVP, H-2D b /EGP and H-2D b /EGS MHC complexes were refolded by dilution. All complexes were isolated following concentration using size-exclusion chromatography. The isolated refolded MHC complexes were thereafter concentrated to 6 mg/ml. Crystals were obtained in hanging drops by vapour diffusion. Crystal screens (Hampton Research, Website, CA, USA) were used to establish initial crystallisation conditions that were then refined in a finer grid.
  • the space group and unit cell parameters were determined using the program XDS, and scaling and reduction of the data were performed using XSCALE.
  • the structure of H-2D b /KVP, H-2D b /EGP and H-2D b /EGS were solved by molecular replacement using the program PHASER.
  • the atomic coordinates of the previously solved crystal structure of H-2D b in complex with the LCMV peptide gp33 (pdb code 1N5A) were used as the search model. Four solutions were unambiguously identified.
  • Further crystallographic refinement was conducted using the program REFMAC5 from the CCP4 package. Five percent of the reflections were set aside for validation and monitoring the refinement by R free •
  • a glycine residue at position 2 and of a proline (or any other aromatic residue, such as a phenylalanine or a tyrosine, or any other cyclic natural or synthetic compound) at position 3 of the peptide might also be responsible for the improved binding of the gplOO peptide with position 2 with a high level of conformational freedom and position 3 aromatic.
  • EGS EGSRNQDWL (SEQ ID NO: 3)
  • EVS EVSRNQDWL (SEQ ID NO: 4)
  • EVP EVPRNQDWL (SEQ ID NO: 5)
  • EGP EGPRNQDWL (SEQ ID NO: 6)
  • KVP KVPRNQDWL (SEQ ID NO: 7)
  • AVP AVPRNQDWL (SEQ ID NO: 9)
  • EGS, EVS, EVP, EGP, KVP, KVA, AVP, KAP and KGP peptides were synthesised in the Leiden University Medical Center. Variants of gpl00 25 - 33 (plp2p3RNQDWL) are designated by the amino acid sequence of position 1, 2 and 3 of the peptide. CpG oligodeoxynucleotides 1826 TCCATGACGTTCCTGACGTT (SEQ ID NO: 24) were synthesised in the Leiden Institute of Chemistry. The following peptides (shown in Table 5 below) were used for testing in the context of HLA-A2 and HLA-AIl and were purchased from the Genscript, USA.
  • GLCTLVAML (SEQ ID NO: 12) is the Epstein-Barr Virus- associated peptide BMLF-I (259-267) .
  • IVTDFSVIK (SEQ ID NO: 16) is the Epstein Barr virus-associated peptide EBNA-4 (416-424) .
  • IGP IGPDFSVIK (SEQ ID NO: 19
  • Binding of peptides to H-2D b was determined using a RMA-S binding assay. Briefly, RMA-S cells were cultured for 2 days at 26°C to achieve expression of ⁇ empty' Class I molecules on the cell surface, Serial dilutions of peptides were added to duplicate cultures of cells to allow for stabilization of MHC molecules. Peptides corresponding to Adenovirus 5 ElA • : 234-243 D b binding epitope SGPSNTPPEI (SEQ ID NO: 21) and MuLV envi 89 -i 96 K b binding epitope SSWDFITV (SEQ ID NO: 22) were used as positive and negative controls, respectively.
  • H-2D b binding index is expressed as (mean fluorescence with peptide - mean fluorescence without peptide) /mean fluorescence without peptide.
  • the results indicate that the combined introduction of a proline at position 3 of the peptide and the use of a glycine at position 2 increased binding affinity of the modified peptides to H-2D b very significantly (by an amplitude not previously observed) . Furthermore, it was found that the increase in binding affinity for MHC-I was achieved without altering the recognition of the peptides by T-cells (as demonstrated by the comparative analysis of the crystal structures of H-2D b /EGS, H-2D b /KVP and H-2D b /EGP) .
  • modified peptides having proline at position 3 or a combination of glycine at position 2 and proline at position 3 is shown with reference to the binding-concentration curves of Figures 4-5 and 12.
  • T2 cells 3 ⁇ lO 5 /well were incubated overnight in 96-well plates with culture medium (a 1:1 mixture of RPMI 1640-Eagle-Hank's amino acid (EHAA) media containing 2.5% FCS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin) with 10 ⁇ g/ml human ⁇ 2-microglobulin (Sigma-Aldrich, St. Louis, MO) and peptides.
  • culture medium a 1:1 mixture of RPMI 1640-Eagle-Hank's amino acid (EHAA) media containing 2.5% FCS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin
  • FI 50 the peptide concentration, ⁇ M, that increases HLA-A2 or HLA-AIl expression by 50% over no peptide control background, was calculated from the titration curve for each peptide .
  • GLP GLPTLVAML ++++ GGP GGPTLVAML HLA-AI l
  • T-cell response assays employed pmel cells specific for H- 2D b /mgpl00 and H-2D b /hgpl00.
  • modified peptides enhanced proliferation of T cells in the draining lymph nodes as well as the non-draining lymph nodes and the spleen ( Figures 9 and 10) .
  • mice Male C57BL/6 mice were obtained from Iffa Credo (France) .
  • gpl00 25 - 33 /D b specific TCR transgenic pmel mice were a kind gift of Dr. N. P. Restifo (Bethesda) and were bred to express the congenic marker
  • mice were housed in the animal facility of the Leiden University Medical Center under specific pathogen free conditions and used between 6 and 12 weeks of age. Experiments were performed in accordance with national legislation and institutional guidelines. In vivo experiments
  • Lymphocytes from spleen and lymph nodes of naive pmel mice were isolated and enriched for T lymphocytes by passing the cell suspension over a nylon wool column.
  • Pmel cells were labelled with 5 ⁇ M CFSE (Molecular Probes, Leiden) and 3 x 10 ⁇ CD8 + T lymphocytes were adoptively transferred by injection into the tail vein.
  • CFSE Molecular Probes, Leiden
  • mice were immunised by subcutaneous injection of a mixture of 50 ⁇ g peptide and 25 ⁇ g CpG in PBS. After 4 days, spleen and draining lymph nodes were removed and single cell suspensions were prepared for analysis.
  • Lymphocytes were incubated with fluorochrome- labelled antibodies (BD biosciences) specific for CD8 (53-6.7) , Thyl.l (HIS51) , and CD62L (MEL-14) .
  • a part of the lymphocytes was incubated for 3 h in the presence of 1 ⁇ g/ml EGS and GolgiPlugTM and thereafter stained for intracellular IFN- ⁇ (XMGl.2) in combination with CD8 and Thyl.l according to the manufacturer's protocols (BD biosciences) .
  • Cells were analyzed using a FACS CaliburTM flow cytometer and Cell Quest Pro software (BD biosciences) .
  • Plots of CFSE, IFN- ⁇ and CD62L are gated on CD8 + , Thyl.l ⁇ live lymphocytes.
  • Immunization assays with mgpl00 25 - 33 peptide EGS (SEQ ID NO: 3) or modified mpgl00 25 - 33 peptide EGP (SEQ ID NO: 6) were carried out using an optimised vaccination protocol.
  • the vaccination protocol involved priming the endogenous gplOO-specific T-cell repertoire. Directly ex vivo no CTL response could be detected, but following in vitro re- stimulation with peptide-loaded dendritic cells it was possible to select murine gplOO specific responses using tetramerical constructs and checking for IFNy expression in the EGP-immunised mice but not in the EGS-immunised group.
  • naive C57BL/6 mice were shaved on the flank and injected subcutaneously on day 0 and day 7 with PBS alone or 50 ⁇ g EGS or 50 ⁇ g EGP peptide.
  • Aldara creme containing 5% imiquimod (3M Health Care Ltd., Leicestershire, UK) was applied to the skin at the injection site.
  • splenocytes were harvested and restimulated in vitro with LPS-matured, irradiated dendritic cells (Dl) loaded with 0.5 ⁇ g/ml EGS.
  • TCR transgenic system The obvious disadvantage of a TCR transgenic system is that the monoclonal CTL population triggered by peptide analogs does not reflect the polyclonal character of endogenous responses.
  • peptide analogs may give rise to CTL responses with different fine-specificities that do not necessarily cross-react with the original peptide target.
  • Endogenous CTL responses against EGS, KVP and the super-peptides EGP and KGP were analyzed in order to address this issue.
  • Groups of C57BL/6 mice were vaccinated with each peptide in combination with the TLR7 ligand imiquimod as an adjuvant (Rechtsteiner et al . , Journal of Immunology, 174, 2476-2480, 2005) .
  • the reactivity of the vaccine-induced CD8 + T-cells isolated from spleens and lymph nodes was analyzed in vitro ( Figure 13A) .
  • Vaccination and in vitro stimulation with EGS did not yield any detectable peptide-specific T-cells while e.g. EGP-induced T-cell responses displayed the most favorable characteristics: they were present at high frequencies and cross-reacted to even very low concentrations of the natural target EGS.
  • Peptide vaccination for treatment of cancer has so far not met clinical success, although vaccine-induced CTL responses can be detected in some patients (Rosenberg et al . , Nature Medicine, 10, 909-915, 2004) .
  • Peptide vaccination can be strongly improved through the use of longer peptides that comprise minimal CTL epitopes in combination with adequate adjuvants that activate the innate immune system (Bijker et al . , Journal of Immunology, 179, 5033-5040, 2007) .
  • the immunotherapeutic potential of the super-peptide EGP was assessed against the aggressive B16 melanoma that expresses the gplOO antigen EGS.
  • the CD8 + T cells raised by EGP efficiently killed B16 melanoma cells in vitro ( Figure 13C) as well as peptide-loaded targets in vivo ( Figure 13D) .
  • the human orthologue KVP performed better than the mouse EGS, but was far less efficient when compared to the analog super- peptide EGP.
  • the induced CTL population displayed a relatively broad repertoire of TCR variable regions that was comparable to that of the other peptide variants ( Figure 14), indicating that super-peptide agonists are different from bacterial superantigens that activate single TCR V ⁇ families (Sundberg et al . , Current Opinion in Immunology, 14, 36-44, 2002) .

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Abstract

Le procédé décrit permet de produire un polypeptide ou un peptide, ou un acide nucléique codant pour le polypeptide ou le peptide. Ledit polypeptide ou peptide comprend une séquence peptidique modifiée ayant une affinité de liaison accrue pour une molécule de la Classe I du CMH et/ou générant une réponse immunitaire en lymphocytes T CD8+ plus élevée. Le procédé comprend les étapes consistant à : identifier une séquence peptidique (« peptide parent ») d'un épitope de lymphocytes T CD8+ d'un antigène, la séquence peptidique pouvant être présentée sur une molécule de Classe I du CMH ; et produire un polypeptide ou un peptide comprenant une séquence peptidique modifiée de l'épitope des lymphocytes T CD8+ ou une molécule d'acide nucléique codant pour ledit polypeptide ou ledit peptide. La séquence peptidique modifiée porte une mutation, par rapport au peptide parent, la mutation introduisant un résidu acide aminé cyclique ou pseudo-cyclique à la position 3 de la séquence peptidique modifiée, définie de telle façon que les résidus sont numérotés séquentiellement, la position 1 correspondant au premier résidu présenté dans la fente de liaison au peptide de la molécule de Classe I du CMH, et le peptide modifié ayant une affinité de liaison plus élevée pour la molécule de Classe I du CMH et/ou générant une réponse en lymphocytes T CD8+ plus élevée, par rapport au peptide parent.
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JP2021506304A (ja) * 2017-12-23 2021-02-22 ルビウス セラピューティクス， インコーポレイテッド 人工抗原提示細胞および使用方法
JP7158483B2 (ja) 2017-12-23 2022-10-21 ルビウス セラピューティクス， インコーポレイテッド 人工抗原提示細胞および使用方法
JP2021508475A (ja) * 2017-12-28 2021-03-11 グリットストーン オンコロジー インコーポレイテッド 共有抗原を標的とする抗原結合タンパク質
CN111886027A (zh) * 2017-12-28 2020-11-03 磨石肿瘤生物技术公司 靶向共同抗原的抗原结合蛋白
CN109293739A (zh) * 2018-01-24 2019-02-01 中国疾病预防控制中心病毒病预防控制所 一种a3超家族通用肿瘤抗原多肽及其应用
EP3935172A4 (fr) * 2019-03-04 2022-12-28 University Health Network Récepteurs de lymphocytes t et leurs procédés d'utilisation
US11733246B2 (en) 2020-11-03 2023-08-22 Indi Molecular, Inc. Compositions, imaging, and therapeutic methods targeting folate receptor 1 (FOLR1)

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