Classifications

• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
  • A61K40/4269—NY-ESO
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/46—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to peptides which are useful in the context of cellular immunology. More particularly, the invention relates to peptides which bind to HLA molecules on the surface of cells. At least some of these peptides also induce the activation of cytolytic T cells, when they are complexed with their partner HLA molecule. Also a part of the invention are the uses of these peptides in areas such as identifying HLA-Cw3 and HLA-Cw6 positive cells, provoking T cells, determining presence of particular T cells, as well as cytolytic T cells themselves.
• T-cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject.
• the family of tum antigen presenting cell lines are immuno genie variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al, J. Exp. Med. 152:1184-1193 (1980), the disclosure of which is incorporated by reference.
• tum antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., "tum + " cells). When these tum + cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "turn-").
• tum + cells When these tum + cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "turn-").
• Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res.43 : 125 (1983). It appears that turn " variants fail to form progressive tumors because they initiate an immune rej ection process.
• mice acquire an immune memory which permits them to resist subsequent challenge to the same turn variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl. Acad. Sci. USA 74:272-275 (1977); Van Pel et al., supra: Uyttenhove et al., supra). Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Nan Pel et al., J. Exp. Med. 157:1992-2001 (1983).
• TRAs tumor rejection antigens
• CTL cytolytic T cell
• a tumor exemplary of the subj ect matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988); Szikora et al, EMBO J. 9:1041-1050 (1990), and Sibille et al., J. Exp. Med. 172:35-45 (1990), the disclosures of which are incorporated by reference.
• the P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line.
• the P815 line has generated many tum variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and PI 98 (Sibille, supra .
• P91A DePlaen, supra
• 35B Stekora, supra
• PI 98 Sudle, supra
• the tum antigens are only present after the tumor cells are mutagenized. Tumor rejection antigens are present on cells of a given tumor without mutagenesis.
• a cell line can be tum + , such as the line referred to as "PI,” and can be provoked to produce tum " variants.
• the anchor residues are positions 5 and 8 of an octamer
• H-2D b they are positions 5 and 9 of a nonapeptide
• the anchor residues for HLA-A1 are positions 3 and 9 of a nonamer.
• positions 2 and 9 are anchors.
• melanoma melanoma antigens which are recognized by cytolytic T cells are now divided into three broad categories.
• the antigens are the expression product of normal genes which are usually silent in normal tissues.
• a second family of melanoma antigens includes antigens which are derived from mutant forms of normal proteins. Examples of this family are MUM-1 (Coulie, et al., Proc. Natl. Acad. Sci. USA 92:7976-7980 (1955)); CDK4 (Wolfel, et al.. Science 269: 1281-1284(1955)): Bcatenin (Robbins, et al., J. Exp. Med. 183 : 1185-1192 (1996)); and HLA-A2 (Brandel, et al., J. Exp. Med. 183:2501-2508 (1996)).
• a third category also discussed, supra, includes the differentiation antigens which are expressed by both melanoma and melanocytes.
• exemplary are tyrosinase, gplOO, gp75, and Melan A/Mart-1.
• tyrosinase gplOO, gp75
• Melan A/Mart-1 exemplary are tyrosinase, gplOO, gp75, and Melan A/Mart-1.
• tyrosinase gplOO, gp75
• Melan A/Mart-1 e.gplOO, gp75
• Melan A/Mart-1 e.gplOO, gp75
• Melan A/Mart-1 e.gplOO, gp75
• Melan A/Mart-1 exemplary are tyrosinase, gplOO, gp75
• Melan A/Mart-1 exemplary are ty
• HLA bindings motif analysis such as that provided by Falk, et al., Nature 357:290-296 (1991) can be very useful in identifying relevant peptides.
• the technique includes inducing a de vovo specific response by naive T cells (Chaux, et al., J. Immunol. 163:2928-2936 (1999); Butterfield, et al., J. Immunol. 161 :5607-5613 (1998)); and in stimulating and expanding in vivo sensitized T cells. See, e.g. Toso, et al Cane. Res. 56:16-20 (1996); Yee, et al, J. Immunol. 157:4079-4086 (1996); Kim, et al, Immunother.20:276-286 (1997): Ferrari, etal.. Blood 90:2406-2416 (1997).
• TheTcells are then used to identify naturally processed tumor peptides eliciting a T cell response.
• HLA-A alleles particularly HLA-A2 presentation.
• MHC/HLA restriction for other MHC/HLA molecules.
• HLA-B27 molecule has been studied most extensively. See, e.g., Parker, et al, J. Immunol. 152 : 163 (1994), incorporated by reference. Its frequency would suggest that, in a given molecule that is processed to MHC/HLA ligands and/or epitopes, HLA-B27 binders might be expected. As will be shown, however, this was not the case with the invention described herein.
• HLA-C molecules and their binding peptides are scant. Binding motifs are not well characterized, and few peptides have been tested. The frequency of HLA-C occurrence is much lower than the occurrence of HLA-A and B molecules, and the HLA-C molecules are far from the first choice for investigation in a population pool.
• One of the unexpected findings of the work described herein was the identification of two HLA-C epitopes, as there was little to suggest these in the literature and, as will be elaborated on herein, from the experimental design.
• the molecule referred to as "NY-ESO-1", as described in, e.g., U.S. Patent No. 5,804,381, incorporated by reference, is recognized as one of the most immunogenic of tumor antigens. Nearly half of patients with advanced cancer express the antigen (Stockert, et al., L Exp. Med. 187:1349-1354 (1998)), and the expression is accompanied by both a strong CD4+ and a strong CD8+ T cell response. See Jager, et al., J. Exp. Med. 191 :625-630 (2000); Jager et al., J. Exp. Med. 167:265-270 (1998); Jager, et al., Proc. Natl. Acad.
• NY-ESO-1 also presents epitopes which bind to HLA-C molecules, such as HLA-Cw3 and HLA-Cw6. See, e.g., p. 7, line 13 after ". . . HLA-Cw3 and HLA-Cw6.”
• NY-ESO-1 has a homologous sequence to another tumor rejection antigen called LAGE-1 (Lethe et al. U.S. Patent No. 5,811,519).
• PBLs Peripheral blood lymphocytes
• RPMI medium 1640 supplemented with 10% human AB serum, L-glutamine (2mM), penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and 1% non essential amino acids.
• the PBLs depleted of CD8+ cells were used as antigen presenting cells ("APCs" hereafter). As will be elaborated in more detail, infra, these cells were either pulsed with lO ⁇ M of peptide, or infected with adenoviral constructs, at 1000 IU/cell, overnight, at 37°C in 300 ⁇ l of serum free medium.
• CD 8 depleted PBLs were secured as described supra. These were then transfected with either adenovirus vectors containing cDNA encoding the NY-ESO- 1 protein, or an "empty" adenoviral vector. PBLs were secured from both a healthy donor, and a cancer patient.
• the resulting shuttle plasmid referred to as pSN2-ICEU-l ⁇ Y-ESO-1 was then digested with ICEU, yielding an expression cassette that contained the CMN promoter/enhancer, ⁇ Y-ESO-1 cD ⁇ A, and a BGH poly A sequence. This fragment was then isolated and cloned into the unique site ICEU-1 site of "pAd Quick" plasmid. This plasmid was then digested with SmaBI, and the digest was used to transfect 293 cells, resulting in a recombinant adenovirus vector that encoded NY-ESO-1.
• PBLs were infected with 1000 IU/cell of the adenoviral constructs, and then incubated overnight at 37°C. Cells were permeabilized, and stained with 1 ⁇ g/ml of monoclonal antibody specific for NY-ESO-1, which is described in Stockert, et al., J. Exp. Med. 187:1349- 1354 (1998), incorporated by reference, and PCT application WO99/53938, both of which are incorporated by reference.
• the CD8 depleted PBLs were then either pulsed with lO ⁇ M samples of SEQ ID NO: 1, transfected with adenovirus encoding NY-ESO-1, as described supra, or with adenovirus encoding green fluorescent protein.
• Autologous CD8+ cells were then stimulated with the PBLs, for eight days. Stimulation was carried out by adding lxl 0 6 APCs per well of CD8+ cells, as described, supra (i.e., adding these to wells containing 5x10 5 CD8+ cells/well). After 8 hours of stimulation, IL-2 was added (10 U/ml), as was IL-7 (20ng/ml). This procedure was repeated, for three days, until cells were harvested for testing.
• Cells were tested in a tetramer assay, by staining the CD8+ T cells in 50 ⁇ l PBS containing 1%FCS, with phycoerythrin ("PE") labeled tetramers. Tetramer synthesis followed Altman, et al., Science 274:94-96 (1996), incorporated by reference. Tetramers were assembled using SEQ ID NO: 1 as the peptide. Cells were stained for 15 minutes at 37°C, after which a commercially available monoclonal antibody specific for CD8, i.e., "Tricolor - CD8mAb" was added, on ice, for 15 minutes. Cells were washed, and analyzed by flow cytometry.
• PE phycoerythrin
• the frequency of tetramer-positive populations were equivalent, i.e., the responses using peptides and adenovirus transfected cells were the same.
• the response was specific for NY-ESO-1, because when adenovirus encoding green fluorescence protein was used, the tetramer staining was negative.
• CD8+ T cells obtained from one subject more fully. Cytospot assays, as described by Jung, etal.,J. Immunol. Meth. 159:197-207 (1993), incorporated by reference and adopted as described herein, were used. CD8+ T cells, presensitized as described, supra, were mixed with autologous, EBV-B target cells, at a 1 :2 ratio, in 300 ⁇ l of serum free medium for 30 minutes. Brefeldin A was added to samples, at lO ⁇ g/ml, for an additional 5 hours. The autologous EBN-B cells, referred to supra, were either pulsed with the peptide of SEQ ID NO: 1, or had been transfected with vaccinia vector constructs.
• the vaccinia virus construct used contained full length NY-ESO-1 cDNA, under the control of vaccinia virus 40K promoter, as taught by Gritz, et al, J. Virol. 64:5948-5957 (1990), incorporated by reference, and E.coli lacZ gene under control of fowlpox virus Cl promoter, as described by Jenkins, et al., AIDS Res. Hum. Retroviruses 7:991-998 (1991), incorporated by reference.
• Foreign sequences were inserted into the thymidine kinase gene of the construct, located in the Hind III region of the genome of vaccinia virus Wyeth strain, following Mazzara, et al., Meth. Enzvmol. 217:557-581 (1993), incorporated by reference.
• CD8+ T cells that were positive to the tetramer described supra were sorted, via flow cytometry using the methods described supra. Two populations were found, i.e., one subpopulation positive to the tetramer, and a second population negative to it.
• Presensitized CD8+ T cells as described supra were added, in quantities ranging from lxlO 3 to 5xl0 4 cells/well, together with 5xl0 4 target cells.
• Target cells were PBLs, pulsed with the peptide of SEQ ID NO: 1, PBLs transfected with vaccinia virus expressing NY-ESO-1, or with EBV-B cells, as described, supra. The cells were incubated for 20 hours in RPMI medium 1640, lacking IL-2 and human serum. Plates were then washed thoroughly with PBS to remove cells, and IFN- ⁇ mAbs (0.2 ⁇ g/ml), were added to the wells.
• the tetramer negative subpopulation was then tested with a panel of EBN-B cells, taken from healthy donors, and transduced with recombinant virus to express ⁇ Y-ESO- 1 but had varying HLA specificities, i.e.:
• LAMPFATPM (SEQ ID NO: 2).
• a patient was selected who was seropositive to NY-ESO-1.
• PBLs were taken from the patient, and following separation of CD8+ T cells as described supra, effector cells were stimulated, in vitro, by CD8 depleted PBLs that had been infected by the adenovirus construct encoding NY-ESO- 1.
• CD8 depleted PBLs that had been infected by the adenovirus construct encoding NY-ESO- 1.
• nearly 40% of sensitized CD8+ T cells were capable of specifically producing IFN- ⁇ in response to NY-ESO- 1 expressed by vaccinia infected, histocompatible EBN B cells.
• HLA-Cw6 was identified as a restriction element for this response, using the EBN-B cells described supra. Anchor motifs for HLA-Cw6 are described by Falk, et al., supra.
• a peptide consisting of amino acids 80-88 was deduced as the relevant nonamer. The peptide was synthesized, tested as described supra, and its recognition by effector cells in an HLA- Cw*0602 restricted fashion was confirmed.
• NY-ESO-1 specific responses were observed against histocompatible EBN-B cells expressing ⁇ Y-ESO-1 via the vaccinia virus vectors. Both the HLA-A2 restricted peptide (SEQ ID NO: 1), and the HLA-Cw3 restricted peptide (SEQ ID NO: 2), were targets of this response. No responses were seen with the seronegative individuals.
• a method for indentifying T cells such as CD8+ T cells which are specific to a peptide/MHC complex, where the peptide derives from a protein of interest.
• a sample believed to contain relevant CD 8+ cells is contacted to an antigen presenting cell, such as a dendritic cell, which has been infected with a first viral vector that encodes the protein of interest.
• an antigen presenting cell such as a dendritic cell
• the CD8+ cells are then contacted with a second population of antigen presenting cells which have been infected with a second viral vector which also encodes the protein of interest, where the second viral vector is different from the first viral vector.
• first viral vector is an adenovirus vector, preferably one that is non replicative
• second vector is a vaccinia vector. It will be understood, however, that these may be reversed, and that only one of these two choices can be used, in combination with a second virus that differs from one of these two choices.
• the method requires an antigen presenting cell, such as a dendritic cell, or some other cell type capable of presenting complexes of an MHC or HLA molecule and a peptide on its surface.
• an antigen presenting cell such as a dendritic cell, or some other cell type capable of presenting complexes of an MHC or HLA molecule and a peptide on its surface.
• the method preferably involves the use of autologous cells, i.e., antigen presenting cells and CD8+ T cells from the same patient, but the methodology can be carried out with allogeneic cells as well.
• Use of the method permits the artisan to identify epitopes that are restricted by their presenting MHC/HLA molecule.
• the method permit identification of peptides which bind to HLA molecules such as HLA-Cw3 and HLA-Cw6 molecules including, but not being limited to the peptides defined by SEQ ID NOS: 2 and 3.
• HLA molecules such as HLA-Cw3 and HLA-Cw6 molecules
• These peptides can be used, e.g., to stimulate production of cytolytic T cells specific for complexes of the HLA molecule and the peptide to identify cells presenting the HLA molecule, and so forth.
• the peptides can be used therapeutically as, e.g., the single peptide component of a formulation designed to enhance an immune response, or as one of a plurality of more than one peptide.
• Such compositions may include an additional component, such as an adjuvant.
• An example of such an adjuvant is GM-CSF, as taught by, e.g., Jager et al., US Patent No. incorporated by reference.
• LAGE LAGE peptides homologous to SEQ ID NOS: 2 and 3, i.e.:
• ITMPFSSPM SEQ ID NO: 4
• a RPDSRLL SEQ ID NO: 5 are also a part of the invention, as epitopes for HLA-Cw3, subtypes HLA-Cw*0303 and HLA- Cw*0304, in particular, and HL-C26, respectively.
• MHC ligands and MHC epitopes.
• these are peptides which bind to MHC molecules, but do not provoke a T cell response when so bound.
• MHC epitopes are peptides which do bind to MHC molecules, and stimulate T cells when confronted with a T cell specific for the peptide/MHC complex.
• Falk et al cited supra, do provide proposed binding motifs for HLA-Cw*3, HLA-Cw*0301, HLA-Cw*0304, HLA-Cw*0601, HLA-Cw*0602. Falk et al do make a distinction between ligands and epitopes, as is evidenced by their paper. It will be seen that no T cell epitopes have been identified for any of these alleles.
• minigenes i.e., nucleic acid molecules consisting of a nucleotide sequence that encodes the peptides of interest.
• the peptides are of a length that permits simple construction of all degenerate sequences which code the epitope of interest.
• These coding sequences can be made a part of an extended "polytopic" sequence, using methods well known in the art, and can be incorporated into coding vectors where the minigene or genes of interest are operably linked to a promoter, for expression in a host cell.
• the minigenes can also be used in concert with genes that encode an MHC molecule of interest, such as HLA-Cw3 or HLA-Cw6 codings sequence.
• the two sequences can constitute part of a single vector, a pair of vectors which are then used in a kit or some other combination that permits the skilled artisan to use them to stimulate a T cell response, and so forth.

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