EP0921805A1 - Peptide, die von melanomspezifischen a1-, a2- und a3-beschränkten zytotoxischen lymphozyten erkannt werden, sowie deren verwendungen - Google Patents

Peptide, die von melanomspezifischen a1-, a2- und a3-beschränkten zytotoxischen lymphozyten erkannt werden, sowie deren verwendungen

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Publication number
EP0921805A1
EP0921805A1 EP97919930A EP97919930A EP0921805A1 EP 0921805 A1 EP0921805 A1 EP 0921805A1 EP 97919930 A EP97919930 A EP 97919930A EP 97919930 A EP97919930 A EP 97919930A EP 0921805 A1 EP0921805 A1 EP 0921805A1
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European Patent Office
Prior art keywords
hla
melanoma
peptide
epitope
ctl
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EP97919930A
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English (en)
French (fr)
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EP0921805A4 (de
Inventor
Craig L. Slingluff
Donald F. Hunt
Jeffrey Shabanowitz
Andrea L. Cox
Victor H. Engelhard
David Kittlesen
J. 36 Binswood Avenue SKIPPER
Ronald C. Hendrikson
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University of Virginia UVA
University of Virginia Patent Foundation
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University of Virginia UVA
University of Virginia Patent Foundation
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Publication of EP0921805A1 publication Critical patent/EP0921805A1/de
Publication of EP0921805A4 publication Critical patent/EP0921805A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • 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
    • A61K39/46—Cellular immunotherapy
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—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
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention is directed to peptides that, in association with Class I MHC molecules, form epitopes recognized by cytotoxic T-cells specific for human melanoma, to immunogens comprising said epitopic peptides, and to related compositions, methods and apparatus. Description of the Background Art
  • Melanoma affects 30,000 new patients per year in the United States. It is a cancer manifested by the unabated proliferation of melanocytes . Eighty percent of melanoma patients are diagnosed during their productive years between the ages of 25 and 65. The incidence of melanoma is rapidly increasing, in 1935 the lifetime risk of developing melanoma was 1:1,500 individuals, at present, the risk has risen to 1:105. It is believed that by the year 2000 the risk of developing melanoma will increase to about 1:70 to 1:90. Early diagnosis and treatment of this disease is crucial.
  • Cytotoxic lymphocyte (CTL) response has been shown to be an important host defense against malignant cells, Rock et al . J . Immunol . , ( 1993 ) , 150 : 1244 .
  • Lymphocytes isolated from patients having melanoma when stimulated in vitro with recombinant interleukin-2 (rIL-2) and autologous melanoma cells, develop a melanoma specific cytotoxic response, Vose et al . , Nature, (1982), 296:359; Knuth et al . , Proc. Natl. Acad. USA, (1984) , 81:3511; Slingluff et al., Arch. Surg., (1987) , 122:1407; Darrow et al., Cancer, (1988) , 62:84; Slingluff et al . , J. Natl.
  • rIL-2 recombinant interleukin-2
  • CTL peripheral blood lymphocytes
  • the evidence that the CTL response to human melanoma is restricted by class I MHC molecules includes demonstration of cross-reactivity for allogenic melanoma cells that share a restricting class I MHC molecule with the autologous tumor.
  • the HLA-A2 molecule and its variants, of which HLA-A2.1 is by far the most common, is an effective restricting element for the melanoma-specific CTL response.
  • melanoma- specific HLA-restricted CTL lyse the majority of A2 + melanomas tested, Darrow et al . , J. Immunol., (1989) , 142:3329; Wolfel et al . , J. Exp.
  • Class I molecules of the Major Histocompatibility Complex bind to peptides derived from intracellular pathogens or from proteins expressed in tumor cells, and present them on the cell surface to the host immune system.
  • the mechanism of peptide presentation involves protein synthesis and proteolysis in the cytosol, followed by transport of peptides into the endoplas ic reticulum (ER) , through the action of the TAP transporter molecules. Peptides then become associated with newly synthesized class 1 molecules, and the resulting complexes move to the cell surface. Proteins that are membrane associated or secreted contain signal sequences that cause them to be contransla- tionally transferred into the ER from membrane-bound ribosomes.
  • MHC Major Histocompatibility Complex
  • An alternate approach to augmenting the T-cell response to melanoma is the use of a vaccine to stimulate CTL in vivo (active specific immunotherapy) .
  • Epitopes for CD8 + CTL are believed to be short, usually 9- residue peptides that bind to a cleft on the surface of the class I MHC molecule, Udaka et al., Cell, (1992) , 69:989; VanBleek et al . , Nature, (1990) , 348:213; Falk et al . , J. Exp. Med., (1991) , 174:425.
  • T cell epitopes are identified by their ability to reconstitute tumor-specific T cell recognition of the transfected cells. The exact T cell epitope is then identified by a combination of molecular subcloning and the use of synthetic peptides based on the predicted amino acid sequence. See, e.g., P. van der Brugge, et al . , Science 254, 1643 (1991) ; C. Traversari, et al . , J. Exp. Med.
  • an HLA-A1 restricted T cell epitope (EADPTGHSY) of a melanoma-associated antigen, MAGE-1 was identified. Traversari, et al . , J. Exp. Med.. 176:1453- 57 (1992) . MAGE-1 is expressed in about 20-40% of cancers of several different tissue types, including melanomas, breast cancers, non-small cell lung cancers, head and neck squamous cell cancers, and bladder cancer. It is also found in the normal male testis.
  • the MAGE gene family also includes another member, MAGE-3, for which a homologous HLA-A1- restricted CTL epitope (EVDPIGHLY) was determined, although only after the first priority date.
  • HLA-A1-restricted CTL epitopes are of limited utility because only a minority of melanomas are HLA-A1 + .
  • the function of the MAGE gene products is not known.
  • the genetic approach has also been used to identify HLA- A2.1-restricted CTL epitopes on tyrosinase. This enzyme is not tumor-specific, ⁇ it is expressed by normal elanocytes as well as melanoma cells.
  • Tyrosinase is involved in melanin biosynthesis.
  • Autologous CTL recognized tyrosinase-derived HLA-A2-restricted epitopes YMNGTMSQV and MLLAVLYCL. See olfel, et al . , Eur. J. Immunol. , 24:759-64 (1994) . However, these peptides were not recognized by the other CTL lines tested.
  • tissue-specific protein is the target of the antibody HMB45, which is specific for melanoma and melanocytes.
  • HMB45 activity Based on the correlation between HMB45 activity and recognition by a single TIL-derived HLA-A2-restricted melanoma-specific CTL line, Bakker, et al . , . Exp. Med. , 179:1005-9 (1994) established that transfection of cells with the gene for gplOO reconstituted the epitope recognized by this T cell.
  • a subsequent study using the same T-cell line to screen transfected cDNA libraries also identified the peptide LLDGTATLRL as being sufficient to reconstitute activity. This study was not published prior to Applicants' first priority date. GplOO is believed to play a role in melanin biosynthesis .
  • AAGIGILTV HLA-A2.1-restricted epitope
  • MART-1 Melan-A
  • Kawakami, et al . , J. Exp. Med.. 180:347-52 (1994) and Proc. Nat. Acad. Sci. USA. 91:3515-19 (1994) and see also Coulie, et al. , J. Exp. Med.. 180:35-42 (1994) .
  • HLA associated peptides have been extracted, isolated and identified from different melanoma lines. These peptides can be used to reconstitute epitopes for HLA-A2.1- and HLA-A3- restricted melanoma- specific CTL. These peptides and the stimulated CTL may be useful for the in vivo immunotherapeutic treatment of melanoma. Aspects of applicants' invention were described in Cox, et al . , Science, 264:716-719 (1994) , which was published on April 29, 1994.
  • the present invention relates to immunogens which are capable of eliciting a melanoma-specific cytotoxic lymphocyte response in at least some individuals, which response is directed to peptide epitopes carried by those immunogens, and to the use of those immunogens in active specific immunotherapy and immunoprophylaxis against melanoma.
  • immunogens may be used as vaccines, in active specific immunotherapy.
  • the immunogens may be administered directly or by gene therapy.
  • the epitopic peptides may also be used to stimulate lymphocytes, the latter then being used for adoptive immunotherapy.
  • a CTL epitope of the present invention is a sequence which is at least substantially homologous with a CTL epitope of the melanoma antigens pMel- 17 and gplOO, (these two antigens are essentially identical) .
  • One such epitope is the peptide 946L.
  • Peptide 9461 is substantially homologous to peptide 946L.
  • a CTL epitope of the present invention is a sequence which is at least substantially homologous with a CTL epitope of tyrosinase.
  • One such epitope is the peptide Lys-Cys-Asp-Ile-Cys-Thr-Asp-Glu-Tyr.
  • Peptides 9461 and 946L, related to a single segment in pMel- 17 (a protein homologous to gplOO) had unexpectedly high A2.1 CTL stimulatory activity. They also are recognized by CTL from different individuals.
  • ALLAVGATK Another pMel-17-derived peptide (ALLAVGATK) had acceptable A3 CTL stimulatory activity, and is the first HLA- A3-associated stimulatory peptide identified in pMel-17 and one of the few, if any, A3-associated peptides identified in melanoma antigens generally.
  • KCDICTDEY is the first Al-restricted epitope to be identified in tyrosinase and one of the few such epitopes identified in melanoma antigens generally (Al epitopes have been identified in MAGE-1 (EADPTGHSY) and MAGE-3 (EVDPIGHLY) ) .
  • a melanoma-specific CTL response from one or more A1-, A2.1- and/or A3- Figure 5.restricted CTLs, and preferably all of them.
  • a melanoma-specific CTL response may be elicited which is restricted by other MHC molecules.
  • FIG. 1A Melanoma specific recognition of autologous tumor by VMM18 CTL.
  • VMM18 cells solid squares
  • K562 open squares
  • VMM12-EBV open circles
  • HLA-A3 " melanoma DM6
  • Figure IB Recognition of VMM18 melanoma by VMM18 CTL was restricted by the class I MHC molecule HLA-A3. Lysis of autologous melanoma was inhibited after incubation of target cells with W6/32 (solid diamonds) and GAP-A3 (solid squares) MAbs, specific for class I MHC and HLA-A3 respectively. Incubation with L243 (open circles) had little effect on recognition of autologous melanoma. Specific lysis of autologous melanoma was 65% (dotted line) , while lysis of VMM12-EBV was 1.5% (solid line) . The effector:target ratio used was 10:1.
  • VMM18 CTL recognize a shared antigen expressed by HLA-A3 + melanomas. Lysis of hot ( 5I chromium labeled) autologous and HLA-A3 + allogeneic melanoma cells (see legend) was inhibited by cold (unlabelled) VMM18 melanoma cells (top fig.) , but not by cold (unlabelled) HLA-A3 ' DM6 melanoma cells (bottom fig.) .
  • FIG. 3 Expression of Pmel-17 reconstitutes recognition of non-melanoma HLA-A3 + target cells by VMM18 CTL.
  • VMM18 CTL lysed 5I Cr-labeled autologous melanoma cells VMM18 (solid squares) as well as a non-melanoma HLA-A3 + cell line VMM12- EBV infected with recombinant vaccinia virus expressing Pmel- 17 (vac-Pmel-17, closed circles) .
  • Minimal lysis of uninfected VMM12-EBV cells (open circles) , or cells infected with control recombinant vaccinia virus expressing influenza nucleoprotein (vac-NP, open triangles) was observed.
  • FIG. 4 Relative ability of Pmel-17 peptides to sensitize non-melanoma target cells for recognition by VMM18 CTL.
  • 51 Cr-labelled T2-A3 cells were incubated with Pmel-17 peptides ALLAVGATK (solid squares) and LLAVGATK (solid triangles) and the control HLA-A3 binding peptide QVPLRPMTYK, from the HIV Nef protein (open circles) .
  • FIGS 5A-B Recognition of autologous and HLA-matched melanomas by melanoma-reactive CTL.
  • VMM12 CTL are evaluated for lysis of a panel of target cells.
  • the VMM12 CTL recognize shared melanoma antigens presented by HLA-Al (VMM15 melanoma cells share HLA-Al with VMM12) , and by HLA-A3 (VMM10 melanoma cells share HLA-A3 with VMM12) .
  • VMM15 CTL are evaluated in the same manner.
  • VMM15 CTL recognize shared melanoma antigens presented by HLA-Al (VMM12 melanoma cells) and by either HLA-Al, -A25, or -B8 (VMM14 melanoma cells) .
  • HLA-A1+ CTL lines recognize tyrosinase peptides on HLA-Al.
  • VMM12 CTL are capable of lysing C1R-A1 cells infected with a vaccinia-tyrosinase construct.
  • VMM15 CTL also recognize tyrosinase.
  • FIGS. 7A-D List of peptides synthesized and tested for recognition by VMM12 and VMM15 CTL. These peptides were predicted from the defined sequence of tyrosinase, accounting for some possible alternate sequences and for possible post- translational modifications. Those listed in the 3rd synthesis were not tested. Figs. 21A-D refers to syntheses 1-4, respectively. Figure 8. VMM15 CTL recognize peptides containing KCDICTDEY in association with HLA-Al. C1R-A1 cells were pulsed with 10 uM, 1 uM and 0.1 uM concentrations of synthetic peptides prior to addition of VMM15 CTL. Background lysis of C1R was approximately 10%.
  • VMM15 CTL recognize a peptide containing KCDICTDEY in association with HLA-Al.
  • C1R-A1 cells were pulsed with peptides at 1 to 0.01 uM concentrations prior to adding VMM12 CTL.
  • the peptides themselves were not cytolytic (open diamonds) .
  • the peptide DAEKCDICTDEY reconstituted an epitope for these VMM12 CTL, although weakly.
  • FIG. 10 Amino acid sequence of tyrosinase, with the position of KCDICTDEY highlighted and underlined. The high proportion of cystine residues and acidic residues are noted relative to the proportion in the intact protein.
  • a melanoma-specific CTL epitope is an epitope which is recognized by a T-cell receptor of at least some cytotoxic lymphocytes of at least some individuals in the population of interest, and which is more frequently or strongly associated with melanoma cells than with at least some other cancer and/or normal cells. There may be some cross-reactivity, for example, with other cells of melanocytic lineage. Absolute specificity is not required, provided that a useful prophy- lactic, therapeutic or diagnostic effect is still obtained.
  • the melanoma-specific CTL epitopes of the present invention are peptides, typically 9-13 amino acids in length, which are sufficiently similar to a melanoma-specific epitope recognized by a melanoma-specific CTL to be useful, under suitable conditions of use, to protect an individual from melanoma, or to be useful in the diagnosis of melanoma or of a patient's ability to fight a melanoma by a CTL response.
  • these epitopes are identical to or otherwise substantially homologous with melanoma-specific peptide epitopes recognized by melanoma-specific CTLs.
  • the family of melanoma epitopes which are recoverable from an individual is dependent on the nature of the binding site of the Class I MHC (HLA) molecules expressed by the individual, and, as a result of the polymorphism of the Class I MHC (HLA) molecules, can vary considerably from one individual to another.
  • the melanoma cell line used as a source of melanoma-specific CTL epitopes may be any melanoma cell line; similarly, the Class I MHC (HLA) molecule may be any such molecule borne by a melanoma which is capable of binding to and presenting a melanoma-specific epitope, including, but not limited to, the various allelic forms of Class I MHC molecules, including but not limited to those enumerated in Table I.
  • the principal genetic loci are denoted as HLA-A, HLA-B, and HLA-C.
  • the preferred epitopic sequence may vary depending on the restriction system.
  • the epitope is one restricted by one of the more prevalent forms (in the melanoma patient population) of these loci.
  • the loci HLA-Al, HLA-A2, HLA-A3 , HLA-B7 and HLA- B8 are of greatest interest.
  • HLA-A2 is of particular interest.
  • the CTL epitopes of the present invention when used in oligopeptide form to reconstitute epitopes for suitable CTL, achieve, at the dosage resulting in maximal lysis of target cells exposed to the stimulated CTL, a percentage lysis of target cells which is at least 10 percentage points higher (more preferably, at least 20 points higher) the background level of lysis of the target cells by the CTLs (i.e., in absence of the peptide) .
  • the peptide concentration at which the epitope-stimulated CTLs achieve half the maximal increase in lysis relative to background is no more than about 1 mM, preferably no more than l ⁇ M, more preferably no more than about 1 nM, still more preferably no more than about 100 pM, most preferably no more than about 10 pM.
  • concentrations of peptide in the pM range are preferably no more than about 1 mM, preferably no more than l ⁇ M, more preferably no more than about 1 nM, still more preferably no more than about 100 pM, most preferably no more than about 10 pM.
  • MAGE-1 peptide EADPTGHSY had half-maximal lysis between 1 and 100 nM (prob about 10) ; while the tyrosinase peptides YMNGTMSQV and MLLAVLYCL reported by Boon induced half-maximal lysis (even with pre-treatment with MA2.1 antibody) at over 10 nM.
  • ALLAVGATK is at present the only pMel-17 derived peptide known to be immunogenic in the context of HLA-A3 , which is expressed by 20% of the patient population. It achieves half-maximal lysis of T2 cells expressing HLA-A3 at a concentration of about 10 nM. While not as potent as our A2.1 peptides, its potency is acceptable.
  • the epitope is recognized by CTLs from at least two different individuals, more preferably at least five different individuals. More preferably, the CTL epitope satisfies two or more of the above desiderata.
  • the 946L peptide although recognized by HLA-A2.1- restricted melanoma-specific CTL, may not oe optimal at present. It is known that some residues on the nonamer peptide are particularly important for binding of the peptide to the MHC molecule (residues 2,9) , while others are particularly important for Tc recognition (residues 4-8) . The other residues may be important for either or both. It is proposed that amino acid substitutions for the 946 peptide may be useful at increasing immunogenicity, particularly by attempting to change residues that may increase binding to the MHC such as changing residue 9 to a valine or residue 3 to anything other than glutamic acid (E) .
  • E glutamic acid
  • epitopes for melanoma-specific CTL these peptides can be screened for their ability to sensitize non-melanoma targets for recognition by melanoma specific CTL. Therefore, in addition to epitopes which are identical to the naturally occurring melanoma-specific epitopes, the present invention embraces epitopes which are substantially homologous with such epitopes, and therefore melanoma- specific in their own right.
  • substantially homologous when used in connection with amino acid sequences, refers to sequences which are substantially identical to or similar in sequence with each other, giving rise to a ho ology in conformation and thus to similar (or improved) biological activity. The term is not intended to imply a common evolution of the sequences.
  • An epitope is considered substantially homologous to a reference epitope if it has at least 10% of an immunological activity of the reference epitope and differs from the reference epitope by no more than one non-conservative substitution not suggested by a known binding motif of the pertinent MHC molecule. Any number of highly conservative, conservative or semi-conservative substitutions, or non- conservative substitutions suggested by known binding motifs, subject to the activity limitation, are permitted.
  • HLA-A specific peptide binding motifs for the HLA molecules Al, A2.1, A3, All and A2 .
  • Engelhard, et al . in Sette, ed. , Naturally Processed Peptides, 57:39-62 (1993) explored the features that determined binding to HLA-A2.1 and HLA-B7. See also Hobohim et al; Eur. J. Immunol., 23:1271-6 (1993) ; Kawakami, et al . , J. Immunol., 154:3961-8 (1995) .
  • the preferred and tolerated AAs for various HLA molecules include (but are not limited to) the following:
  • YLEPGPVTV An example of a peptide variant which satisfies the known binding motif is YLEPGPVTV. This differs from 946L at position 9. However, V is a preferred a.a. at position 9 of HLA-A2.1 binding peptides.
  • Multiple mutagenesis may be used to screen a few residue positions intensely or a larger number of positions more diffusely.
  • One approach is to explore at least a representative member of each a.a. type at each position, e.g., one representative of each of exchange groups I-V as hereafter defined.
  • Gly and Pro are screened in addition to one other group I residue.
  • at least one screened residue is an H-bonding resiude. If a positive mutant features a particular representative, like amino acids can be explored in a subsequent library. If, for example, a Phe substitution improves binding, Tyr and Trp can be examined in the next round.
  • Asp/Glu and Gln/Asn sets can be merged. It is known from comparison of peptide 1030 with the homologous tyrosinase segment that substitution of Asn for Asp in position 3 reduces CTL activity 100-fold. However, a multiple mutagenesis strategy could identify compensating mutations at other sites.
  • Met Gly Gly These strategies take into account both conservative substitutions for the wild type AAs, and the known Al, A2.1 and A3 binding motifs.
  • residues to vary may also make comparisons of the sequences of the naturally processed MHC associated peptides, and may obtain 3D structures of the MHC: peptide: TCR complexes, in order to identify residues involved in MHC or TCR binding. Such residues may either be left alone, or judiciously mutated in an attempt to enhance MHC or TCR binding.
  • substantially homologous epitopes it is also possible to predict substantially homologous epitopes by taking into account studies of sequence variations in families of naturally occurring homologous proteins. Certain amino acid substitutions are more often tolerated than others, and these are often correlatable with similarities in size, charge, etc. between the original amino acid and its replacement. Insertions or deletions of amino acids may also be made. N- and C-terminal truncations or extensions are more likely to be tolerated than internal deletions or insertions. With regard to truncation, the peptide may be truncated by one or more amino acids and still be substantially homologous, however, it cannot be fewer than five amino acids. Extensions are permissible, however, note that larger peptides are digested in vivo prior to presentation.
  • Conservative substitutions may be made in the amino acid sequence of the proteins of interest without compromising the desired properties of the peptides, i.e., induction of cytotoxic T-lymphocytes in a patient when administered thereto.
  • Semi-conservative substitutions are defined to be exchanges between two of groups (I)-(V) above which are limited to supergroup (A) , comprising (I) , (II) and (III) above, or to supergroup (B) , comprising (IV) an (V) above. Also, Ala is considered a semi-conservative substitution for all non group I amino acids.
  • substitutions are not limited to the genetically encoded, or even the naturally occurring amino acids.
  • the desired amino acid may be used directly.
  • a genetical ⁇ ly encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues .
  • the following examples of chemical derivatives are provided by way of illustration and not by way of limitation.
  • Aromatic amino acids may be replaced with D- or L-naphylalanine, D- or L-Phenylglycine, D- or L-2-thieney- lalanine, D- or L-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- or L- (2-pyridinyl) -alanine, D- or L- (3-pyridinyl) -alanine, D- or L- (2-pyrazinyl) -alanine, D- or L- (4-isopropyl) -phenylglycine, D- (trifluoromethyl) - phenyl- glycine, D- (trifluoromethyl) -phenylalanine, D-p-fluoro- phenylalanine, D- or L-p-biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or L
  • Acidic amino acids can be substituted with non- carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono) -alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfated (e.g., -S0 3 H) threonine, serine, tyrosine.
  • (phosphono) -alanine glycine, leucine, isoleucine, threonine, or serine
  • sulfated e.g., -S0 3 H
  • substitutions may include unnatural hyroxylated amino acids made by combining "alkyl” (as defined and exemplified herein) with any natural amino acid.
  • Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino) -acetic acid, or other (guanidino) alkyl-acetic acids, where "alkyl” is define as above.
  • Nitrile derivatives e.g., containing the CN-moiety in place of COOH
  • methionine sulfoxide may be substituted for methionine. Methods of preparation of such peptide derivatives are well known to one skilled in the art.
  • Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • any amino acid can be replaced by the same amino acid but of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S configuration, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D- amino acid but which can additionally be referred to as the R- or the S-, depending upon its composition and chemical configuration.
  • Such derivatives have the property of greatly increased stability to degradation by enzymes, and therefore are advantageous in the formulation of compounds which may have longer in vivo half lives, when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • the thiol group of cysteine reacts very rapidly with alkyl halides, such as iodoacetate, iodoacetamide, methyl iodine, and so on, to give the corresponding stable alkyl (substituted or unsubstituted) derivatives, such as -CH 2 -S- CH 3 .
  • alkyl halides such as iodoacetate, iodoacetamide, methyl iodine, and so on
  • alkyl halides such as iodoacetate, iodoacetamide, methyl iodine, and so on
  • alkyl halides such as iodoacetate, iodoacetamide, methyl iodine, and so on
  • the thiol group can also add across double bonds such as those of N-ethylmaleimide or of maleic anhydride, and it can open the ring of ethyleneimine
  • Thiols form complexes with various metal (especially mercury, silver, arsenic, copper, iron, zinc, cobalt, molybdenum, manganese and cadmium ions) and organometal ions (e.g., R-Hg + , such as para-mercuribenzoic acid) .
  • metal especially mercury, silver, arsenic, copper, iron, zinc, cobalt, molybdenum, manganese and cadmium ions
  • organometal ions e.g., R-Hg + , such as para-mercuribenzoic acid
  • the thiol group may be oxidized to yield a disulfide bond or a sulfonate.
  • a thiol may be converted to a disulfide by thiol-disulfide exchange, for example, exchange with an aromatic disulfide such as di hionitrobenzoic acid (DTNB) or Ellman's reagnet .
  • DTNB di hionitrobenzoic acid
  • Ellman's reagnet Ellman's reagnet
  • a cysteine residue may be disulfide bonded to a cysteine residue in the same or a different peptide, or to a free cysteine.
  • cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines) , such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo- beta- (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.
  • compounds such as bromotrifluoroacetone, alpha-bromo- beta- (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitro
  • Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alp- ha-amino-containing residues include compounds such as imidoesters/e.g. , as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues per se The specific modification of tyrosyl residues per se is well-known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups may be selectively modified by reaction with carbodiimides (R' -N-C-N-R 1 ) such as l-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1- ethyl-3- (4-azonia-4,4- dimethylpentyl) carbodiimide.
  • carbodiimides R' -N-C-N-R 1
  • aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues may be readily deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.
  • Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers, according to known method steps.
  • Commonly used cross-linking agents include, e.g., 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, in- eluding disuccinimidyl esters such as 3,3'- dithiobis (suc- cinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1, 8-octane .
  • Derivatizing agents such as methyl-3- [ (p-azidophenyl) dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent NOS. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 (which are herein incorporated entirely by reference) , may be employed for protein immobilization.
  • Derivatized moieties may impart altered affinity for their target, altered immunogenicity, or improved solubility, absorption, biological half life, and the like, or attenuated undesirable side effects. Moieties capable of mediating such effects are disclosed, for example, in Remington' s Pharmaceutical Sciences, 16th ed. , Mack Publishing Co . , Easton, PA (1980) .
  • Modifications are not limited to the side chains of the amino acids.
  • the peptides may also comprise isoteres of two or more residues in the immunogenic peptide.
  • An isotere as defined here is a sequence of two or more residues that can be sustituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence.
  • the term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally. Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983) .
  • the melanoma-specific immunogen of the present invention is a molecule corresponding to or otherwise comprising a melanoma-specific CTL epitope as previously described.
  • the immunogen may comprise one or more melanoma-specific CTL epitopes, which may be the same or different.
  • the immunogen is chosen so that at least one epitope is effective in each of two or more restriction systems, e.g., HLA-Al and HLA-A3; HLA-Al and HLA-A2; HLA-A2 and HLA-A3; and HLA-Al, -A2 and -A3.
  • a single epitope may be effective in more than one restriction system.
  • HLA-A2 and HLA-69, or HLA-A3 and HLA-A11 are pairs of MHC molecules having similar peptide binding motifs.
  • two or more epitopes at least one for each MHC molecule of interest will need to be provided. These epitopes may be separate or overlapping.
  • compositions of the present invention may include two or more immunogens which present different epitopes.
  • the immunogen comprises a plurality of such epitopes, they may be linked directly, or through a spacer of some kind, or by noncovalent means such as an avidin:biotin complex.
  • the immunogen may take any form that is capable of eliciting a melanoma-specific cytotoxic immune response.
  • the immunogen may be a fusion of a plurality of CTL epitopes which is sufficiently large to be immunogenic, a conjugate of one or more epitopes to a soluble immunogenic macromolecular carrier, such as serum albumin, keyhole limpet hemocyanin, or dextran, a recombinant virus engineered to display the epitope on its surface, or a conjugate of a plurality of epitopes to a branched lysine core structure, a so-called "multiple antigenic peptide" (see Posnett, et al . , J. Biol . Chem., 263 :1719-25, 1988) .
  • a soluble immunogenic macromolecular carrier such as serum albumin, keyhole limpet hemocyanin, or dextran
  • a recombinant virus engineered to display the epitope on its surface or a conjugate of a plurality of epitopes to a branched lysine core structure,
  • the immunogenic conjugate may also comprise moieties intended to enhance the immune response, such as a T helper peptide, a cytokine or an adjuvant; a targeting agent, such as an antibody or receptor ligand or ligand analogue; or a stabilizing agent, such as a lipid.
  • moieties intended to enhance the immune response such as a T helper peptide, a cytokine or an adjuvant
  • a targeting agent such as an antibody or receptor ligand or ligand analogue
  • a stabilizing agent such as a lipid.
  • the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues.
  • the CTL peptide may be linked to the T helper peptide without a spacer.
  • the immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide.
  • the amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
  • the immunogen may present one or more such epitopes already known in the art, such as the following:
  • Isoleucine (I) at position 5 is the result of mutation.
  • the wild type sequence si EEKLSWLF. 6 Phenylalanine (F) at pos. 9 is the result of mutation.
  • the wild type sequence is SYLDSGIHS.
  • CTL epitopes may be presented on two or more different immunogens. These may be administered separately, or as part of a mixture, e.g., a mixture of epitopic peptides.
  • the peptide portion of the immunogens of the present invention may be produced by any conventional technique, including
  • the peptides disclosed herein may be produced, recombinantly, in a suitable host, such as bacteria from the genera Bacillus, Escherichia, Salmonella, Erwinia, and yeasts from the genera Hansenula, Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces, or cultured mammalian cells such as COS-1.
  • a suitable host such as bacteria from the genera Bacillus, Escherichia, Salmonella, Erwinia, and yeasts from the genera Hansenula, Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces, or cultured mammalian cells such as COS-1.
  • the more preferred hosts are microorganisms of the species Pi chia pas tori s , Bacillus subtilis, Bacillus brevis, Saccharomyces cerevi ⁇ iae, Escherichia
  • Any promoter, regulatable or constitutive, which is functional in the host may be used to control gene expression. It has been found that peptide fragments from the protein pMEL17 reconstitute HLA A2.1 and A3 epitopes.
  • the pMEL17 gene is a single-stranded cDNA reading 5' to 3 ' .
  • the gene encoding for pMEL17 is: GGAAGAACAC AATGGATCTG GTGCTAAAAA GATGCCTTCT TCATTTGGCT GTGATAGGTG CTTTGCTGGC TGTGGGGGCT ACAAAAGTAC CCAGAAACCA GGACTGGCTT GGTGTCTCAA GGCAACTCAG AACCAAAGCC TGGAACAGGC AGCTGTATCC AGAGTGGACA GAAGCCCAGA GACTTGACTG CTGGAGAGGT GGTCAAGTGT CCCTCAAGGT CAGTAATGAT GGGCCTACAC TGATTGGTGC AAATGCCTCC TTCTCTATTG CCTTGAACTT CCCTGGAAGC CAAAAGGTAT TGCCAGATGG GCAGGTTATC TGGGTCAACA ATACCATCAT CAATGGGAGC CAGGTGTGGG GAGGACAGCC AGTGTATCCC CAGGAAACTG ACGATGCCTG CATCTTCCCT GATGGTGGAC CTTGCCCATC TGGCTCTTGG TCTC
  • the peptide 946L YLEPGPVTA reconstitutes an A2.1 epitope. Its native encoding gene sequence is TAC CTG GAG CCT GGC CAA GTC ACT GCC. Because this peptide has proven immunologic activity, it is ideal for specific immunization. Such immunization may be accomplished either directly, or by use of a vaccine consisting of virus (e.g., Vaccinia) encoding or HLA-A2 cells expressing a genetic sequence encoding this peptide. -The peptide ALLAVGATK, which corresponds to residues 17-25 of pMel-17, reconstitutes an A3 epitope.
  • virus e.g., Vaccinia
  • tyrosinase- related peptide 1030 YMDGTMSQV
  • TAT ATG GAT GGA ACA ATG TCC GAG GTA which reconstitutes an A2- epitope
  • KCDICTDEY which reconstitutes an Al epitope of tyrosinase.
  • the Genetic Code can readily be used to design a gene encoding an arbitrary amino acid sequence, such as that of the preferred HLA-Al epitope, KCDICTDEY, or the preferred HLA-A3 epitope, ALLAVGATK.
  • codon preferences of the intended host organism Preferably, where more than one codon could be used to encode a particular amino acid, consideration is given to the codon preferences of the intended host organism.
  • sequences may be constructed in such a manner, including the appropriate expression systems for use in gene therapy procedures. Because several different nucleotide sequences may encode a single amino acid, alternate DNA sequences may also encode these peptides.
  • Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D., et al., Molecular Biology of the Gene. Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987) ; Darnell, J.E., et al . , Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986) ; Lewin, B.M., Genes II. John Wiley & Sons, publishers, New York, N.Y. (1985) ; Old, R.W. , et al .
  • Chemical Peptide Synthesis is a rapidly evolving area in the art, and methods of solid phase peptide synthesis are well-described in the following references, hereby entirely incorporated by reference: (Merrifield, B., J. A er. Chem. Soc. 85:2149-2154 (1963) ; Merrifield, B., Science 232:341-347 (1986) ; Wade, J.D., et al., Biopolvmers 25:S21-S37 (1986) ; Fields, G.B., Int. J. Polypeptide Prot. Res. 3 . 5:161 (1990) ; MilliGen Report Nos. 2 and 2a, Millipore Corporation, Bedford, MA, 1987) Ausubel, et al, supra. and Sambrook, et al, supra.
  • such methods involve blocking or protecting reactive functional groups, such as free amino, carboxyl and thio groups. After polypeptide bond formation, the protective groups are removed (or de-protect- ed) . Thus, the addition of each amino acid residue requires several reaction steps for protecting and deprotecting.
  • Current methods utilize solid phase synthesis, wherein the C-terminal amino acid is covalently linked to an insoluble resin particle large enough to be separated from the fluid phase by filtration. Thus, reactants are removed by washing the resin particles with appropriate solvents using an automated programmed machine. The completed polypeptide chain is cleaved from the resin by a reaction which does not affect polypeptide bonds.
  • tBoc method the amino group of the amino acid being added to the resin-bound C-terminal amino acid is blocked with tert-butyloxycarbonyl chloride (tBoc) .
  • This protected amino acid is reacted with the bound amino acid in the presence of the condensing agent dicyclohexylcarbodiimide, allowing its carboxyl group to form a polypeptide bond the free amino group of the bound amino acid.
  • the amino-blocking group is then removed by acidification with trifluoroacetic acid
  • TFA gaseous carbon dioxide
  • isobutylene isobutylene
  • HF hydrogen fluoride
  • trifluoro- methanesulfonyl derivatives is common at the end of the synthesis to cleave the benzyl-derived side chain protecting groups and the polypeptide-resin bond.
  • the preferred "Fmoc” technique has been introduced as an alternative synthetic approach, offering milder reaction conditions, simpler activation procedures and compatibility with continuous flow techniques.
  • This method was used, e.g., to prepare the peptide sequences disclosed in the present application.
  • the oc-amino group is protected by the base labile 9-fluorenylmethoxycarbonyl (Fmoc) group.
  • the benzyl side chain protecting groups are replaced by the more acid labile t-butyl derivatives.
  • Repetitive acid treatments are replaced by deprotection with mild base solutions, e.g., 20% piperidine in dimethyl- formamide (DMF) , and the final HF cleavage treatment is eliminated.
  • a TFA solution is used instead to cleave side chain protecting groups and the peptide resin linkage simultaneously.
  • At least three different peptide-resin linkage agents can be used: substituted benzyl alcohol derivatives that can be cleaved with 95% TFA to produce a peptide acid, methanolic ammonia to produce a peptide amide, or 1% TFA to produce a protected peptide which can then be used in fragment condensation procedures, as described by Atherton, E., et al., J. Chem. Soc. Perkin Trans. 1:538-546 (1981) and Sheppard, R.C., et al . , Int. J. Polypeptide Prot . Res . 2J): 51-454 (1982) .
  • highly reactive Fmoc amino acids are available as pentafluorophenyl esters or dihydro- oxobenzotriazine esters derivatives, saving the step of activation used in the tBoc method.
  • the preferred animal subject of the present invention is a primate mammal.
  • mammal an individual belonging to the class Mammalia, which, of course, includes humans.
  • the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.
  • non-human primate is intended any member of the suborder Anthropoidea except for the family Hominidae.
  • Such non-human primates include the superfamily Ceboidea, family Cebidae (the New World monkeys including the capuchins, howlers, spider monkeys and squirrel monkeys) and family Callithricidae (including the marmosets) ; the superfamily Cercopithecoidea, family Cercopithecidae (including the macaques, mandrills, baboons, proboscis monkeys, mona monkeys, and the sacred hunaman monkeys of India) ; and superfamily Hominoidae, family Pongidae (including gibbons, orangutans, gorillas, and chimpanzees) .
  • the rhesus monkey is one member of the macaques.
  • protection is intended to include “prevention,” “suppression” and “treatment.”
  • Prevention involves administration of the protein prior to the induction of the disease.
  • Secondary involves administration of the composition prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after the appearance of the disease.
  • composition may be administered parentally or orally, and, if parentally, either systemically or topically.
  • Parenteral routes include subcutaneous, intravenous intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. One or more such routes may be employed.
  • Parenteral administration can be, .e.g., by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.
  • the immunization is preferably accomplished initially by intramuscular injection followed by intradermal injection, although any combination of intradermal and intramuscular injections may be used.
  • the suitable dosage of a immunogen of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight .
  • a drug Prior to use in humans, a drug will first be evaluated for safety and efficacy in laboratory animals.
  • the total dose required for each treatment may be administered in multiple doses (which may be the same or different) or in a single dose, according to an immunization schedule, which may be predetermined or ad hoc.
  • the schedule is selected so as to be immunologically effective, i.e., so as to be sufficient to elicit an effective CTL response to the antigen and thereby, possibly in conjunction with other agents, to provide protection.
  • Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization
  • peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.
  • the doses may be given at any intervals which are effective. If the interval is too short, immunoparalysis or other adverse effects can occur. If the interval is too long, immunity may suffer. The optimum interval may be longer if the individual doses are larger. Typical intervals are 1 week, 2 weeks, 4 weeks (or one month) , 6 weeks, 8 weeks (or two months) and one year. The appropriateness of administering additional doses, and of increasing or decreasing the interval, may be reevaluated on a continuing basis, in view of the patient's immunocompetence (e.g., the level of antibodies to melanoma-associated antigens) .
  • immunocompetence e.g., the level of antibodies to melanoma-associated antigens
  • the concentration of CTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the immunogen is dissolved or suspended in an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, as well as increase the half-life of the peptide composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among melanocytes or melanomas, or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired peptide of the invention can be directed to the site of target cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. , Ann. Rev. Biophvs . Bioeng. 9:467 (1980) , U.S. Patent Nos . 4,235,871, 4,501,728, 4,837,028, and 5,019369, incorporated herein by reference.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired melanoma cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • each vaccine preparation will include 1-100 ⁇ g of the peptide epitope.
  • the composition may also include an adjuvant. Typical adjuvants include proteins, peptides, carbohydrates, lipids and liposaccharides.
  • DETOX Riv Im unochemicals
  • Other adjuvants include QS-21, Montanide ISA-21, incomplete
  • Montanide ISA- 51 is manufactured by Seppic, Inc. (75 Quai D'Orsay, 75321, Paris, France) . Its composition is manide oleate in mineral oil solution.
  • QS-21 is manufactured by Cambridge Biotech (365 Plantation Street, Worcester, MA 01605-2376) . It is a triterpene glycoside isolated from the bark of a South American tree (Quillaja saponaria) .
  • the tradename for QS-21 is StimulonTM. Its molecular formula is C 92 0 46 H 148 , and its molecular weight is 1,990. Its complete chemical name is
  • the adjuvant may be conjugated to the epitope and not simply a part of a mixture. See Deres, et al, Nature, 342:561-4 (1989) .
  • the composition may also include an immunomodulator, especially cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, Interferon-alpha, Interferon-gamma, Granulocyte Macrophage Colony Stimulating Factor (GMCSF) , Tumor Necrosis Factor (TNF) -alpha, and TNF- beta.
  • an immunomodulator especially cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, Interferon-alpha, Interferon-gamma, Granulocyte Macrophage Colony Stimulating Factor (GMCSF) , Tumor Necrosis Factor (TNF) -alpha, and TNF- beta.
  • cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-12
  • Interferon-alpha Interferon-gamma
  • GMCSF Granulocyte
  • the composition may also include antigen-presenting cells, such as dendritic cells or macrophages.
  • antigen-presenting cells such as dendritic cells or macrophages.
  • the APCs are harvested, e.g., from peripheral blood or bone marrow, conjugated, covalently or noncovalently (e.g., by pulsing) to the immunogen, e.g., a peptide, and administered to the patient.
  • composition may also include a molecule which activates or helps in activating CTLs, such as a CD-28 stimulatory molecule (e.g., B7.1, B7.2, or anti-CD28) . If the molecule may be administered in place of the molecule itself.
  • a CD-28 stimulatory molecule e.g., B7.1, B7.2, or anti-CD28
  • CD80 (B7 BB1) is expressed on activated B cells and dendritic cells. It is a ligand for CD28 and CTLA-4. It has been found to represent two (partially homologous) proteins, B7-1 and B7-2. See Ramarathinam, et al . T cell costimulation by B7/BB1 induces CD8 T-cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells. J.Exp. Med. 1994: 1790: 1205-1214; Freeman et al . Cloning of B7-2: a CTLA-4 counter-receptor that costi ulates human T cell proliferation.
  • a pharmaceutical composition according to the present invention may further comprise at least one cancer chemo ⁇ therapeutic compound, such as one selected from the group consisting of an anti-metabolite, a bleomycin peptide antibiotic, a podophyllin alkaloid, a Vinca alkaloid, an alkylating agent, an antibiotic, cisplatin, or a nitrosourea.
  • at least one cancer chemo ⁇ therapeutic compound such as one selected from the group consisting of an anti-metabolite, a bleomycin peptide antibiotic, a podophyllin alkaloid, a Vinca alkaloid, an alkylating agent, an antibiotic, cisplatin, or a nitrosourea.
  • a pharmaceutical composition according to the present invention may further or additionally comprise at least one viral chemotherapeutic compound selected from gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon- ⁇ , interferon-j0, interferon- ⁇ , thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxy- nucleosides, or ganciclovir. See, e.g., Katzung, supra , and the references cited therein on pages 798-800 and 680-681, respectively, which references are herein entirely incorporated by reference.
  • at least one viral chemotherapeutic compound selected from gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon- ⁇ , interferon-j0, interferon-
  • the pharmaceutical composition may instead comprise a vector comprising an expressible gene encoding such an immunogen.
  • the pharmaceutical composition and method would then be chosen so that the vector was delivered to suitable cells of the subject, so that the gene would be expressed and the immunogen produced in such a manner as to elicit an immune response.
  • a preferred vector would be a Vaccinia virus, such as a construct containing a minigene encoding the peptide
  • a gene encoding the protein pMel-17 is also of some interest. In the case of genes encoding naturally occurring proteins, or peptide fragments thereof, one may, but need not, use the DNA sequence which encodes the proteins or peptides in nature. A preferred route for immunization would be scarification. A preferred immunization protocol would be 10E6 to 10E8 pfu/dose in the initial injection, followed up with boosters at 1,3 and 12 months. The boosters could be the previously described immunogen-containing composition.
  • vaccinia virus constructs have been used for immunization against hepatitis B (Moss, et al. , Nature, 311, 67, 1984) , herpes simplex virus (Wacchsman, et al .. Biosci . Rep. 8., 323; 334, 1988) , parainfluenza type 3 (Spriggs, et al.. , J. Virol., 6_2, 1293, 1988) , and Lassa fever virus
  • Vaccinia virus constructs comprising gene for cancer- associated antigens have also been prepared (Lathe, et al . , Nature, 326, 878, 1987; Bernards, et al .. Proc. Natl . Acad. Sci. USA, 84 , 6854, 1987; Estin, et al . , Proc. Natl. Acad. Sci. USA. 85, 1052, 1988) .
  • the composition may comprise melanoma-specific CTL.
  • Antigenic peptides may be used to elicit CTL ex vivo. Ex vivo CTL responses to a melanoma antigen are induced by incubating in tissue culture the patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-8 weeks) , in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell. In order to optimize the in vi tro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells may be maintained in an appropriate serum-free medium.
  • an amount of antigenic peptide is added to the stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the stimulator cells.
  • a sufficient amount of peptide is an amount that will allow about 200, and preferably 200 or more, human Class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell.
  • the stimulator cells are incubated with at least 1 mg/ml, more preferably >20 ⁇ g/ml peptide.
  • Resting or precursor CD8+ cells are then incubated in culture with the appropriate stimulator cells for a time period sufficient to activate the CD8+ cells.
  • the CD8+ cells are activated in an antigen-specific manner.
  • the ratio of resting or precursor CD8+ (effector) cells to stimulator cells may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the within-described treatment modality is used.
  • the lymphocyte :stimulator cell ratio is in the range of about 1:5 to 20:1, more preferably 3:1 to 5:1.
  • the effector/stimulator culture may be maintained for as long a time as is necessary to stimulate a therapeutically useful or effective number of CD8+ cells.
  • the induction of CTL in vi tro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC.
  • the number of specific MHC/peptide complexes per APC is crucial for the stimulation of CTL, particularly in primary immune responses. While small amounts of peptide/MHC complexes per cell are sufficient to render a cell susceptible to lysis by CTL, or to stimulate a secondary CTL response, the successful activation of a CTL precursor (pCTL) during primary response requires a significantly higher number of MHC/peptide complexes.
  • Peptide loading of empty major histocompatability complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses. Often it is useful, in the generation of peptide- specific CTL, to stimulate with mutant cell lines that have some empty MHC molecules.
  • An exmample is the human lymphoid cell line, T2.
  • mutant cell lines expressing every MHC molecule are not yet available. Thus, in some cases, it may be useful to strip endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest.
  • non-transformed (non-tumorigenic) , non- infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies.
  • This application discloses methods for stripping the endogenous MHC-associated peptides from the surface of APC followed by the loading of desired peptides.
  • a stable MHC class I molecule is a trimeric complex formed of the following elements: 1) a peptide usually of 8 - 10 residues, 2) a transmembrane heavy polymorphic protein chain which bears the peptide-binding site in its ⁇ l and c.2 domains, and 3) a non-covalently associated non-polymorphic light chain, /3 2 microglobulin. Removing the bound peptides and/or dissociating the j3 2 microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to MHC class I molecules on the APC without causing their degradation before exogenous peptides can be added to them.
  • Two possible ways to free up MHC class I molecules of bound peptides include the culture temperature from 37°C to 26°C overnight to destabilize /3 2 microglobulin and stripping the endogenous peptides from the cell using a mild acid treatment.
  • the methods release previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to the empty class I molecules.
  • the cold- temperature incubation method enables exogenous peptides to bind efficiently to the MHC complex, but requires an overnight incubation at 26°C which may slow the cell's metabolic rate. It is also likely that cells not actively synthesizing MHC molecules (e.g., resting PBMC) would not produce high amounts of empty surface MHC molecules by the cold temperature procedure .
  • Harsh acid stripping involves extraction of the peptides with trifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinity purified class I-peptide complexes. These methods are not feasible for CTL induction, since it is important to remove the endogenous peptides while preserving APC viability and an optimal metabolic state which is critical for antigen presentation.
  • Mild acid solutions of pH 3 such as glycine or citrate-phosphate buffers have been used to identify endogenous peptides and to identify tumor associated T cell epitopes. The treatment is especially effective, in that only the MHC class I molecules are destabilized (and associated peptides released) , while other surface antigens remain intact, including MHC class II molecules.
  • Activated CD8+ cells may be effectively separated from the stimulator cells using one of a variety of known methods.
  • monoclonal antibodies specific for the stimulator cells, for the peptides loaded onto the stimulator cells, or for the CD8+ cells (or a segment thereof) may be utilized to bind their appropriate complementary ligand.
  • Antibody-tagged molecules may then be extracted from the stimulator-effector cell admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.
  • Effective, cytotoxic amounts of the activated CD8+ cells can vary between in vi tro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 X 10 6 to about 1 X 10 12 , more preferably about 1 X 10 8 to about 1 X 10 11 , and even more preferably, about 1 X 10 9 to about 1 X 10 10 activated CD8+ cells are utilized for adult humans, compared to about 5 X 10 6 - 5 X 10 7 cells are used in mice.
  • the activated CD8+ cells are harvested from the cell culture prior to administration of the CD8+ cells to the individual being treated. It is important to note, however, that unlike other present and proposed treatment modalities, the present method preferably uses a cell culture system that is not tumorigenic .
  • Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Patent No. 4,844,893 to Honsik, et al. and U.S. Patent No. 4,690,915 to Rosenberg.
  • administration of activated CD8+ cells via intravenous infusion is appropriate.
  • HLA-A2.1+ or HLA-A3+ cells autologous B cells, macrophages, or dendritic cells, ideally
  • peptide e.g., peptide 946, YXEPGPVTA
  • the patients could be pre-stimulated with a peptide vaccine prior lymphocyte harvest if the existing response was inadequate.
  • Lymphocytes stimulated with peptide in vitro could then be expanded to 10 10 or 10" cells, then re-infused into the patients in a manner analogous to that used for LAK cell therapy. It is expected that the adoptively transferred CTL would survive best with IL-2 infusion at low to intermediate doses, and that putative inhibitors of Tc suppression (eg: cyclophosphamide) may be employed also, prior to the infusions of CTL.
  • putative inhibitors of Tc suppression eg: cyclophosphamide
  • TIL tumor infiltrating lymphocytes
  • a melanoma-specific diagnostic agent is (1) a molecule which is or which comprises a melanoma-specific epitope as previously defined, and which is labeled, immobilized, or otherwise rendered suitable for diagnostic use, or (2) an antibody which specifically binds such a melanoma-specific epitope, and which is labeled, immobilized, or otherwise rendered suitable for diagnostic use, or (3) a T-cell line (e.g., murine or human) , which specifically recognizes a melanoma-specific epitope.
  • a T-cell line e.g., murine or human
  • the relationship between the host's immune response and his or her tumor is poorly understood. Better understanding of that response depends on evaluation of the specific responses against individual epitopes, such as the 946 peptide. If patients do have an immune response to 946 naturally, then evaluation and quantitation of that by precursor frequency analysis of the CTL in the patient's blood pool may permit some assessment of the protection that person's immune system is providing. As new therapies become available for melanoma, it may be useful to screen patients for the presence of the 946 peptide on their tumor and the presence of CTL in their blood pool with specificity for the 946 peptide on HLA-A2.
  • the peptides of the present invention may be used to screen a sample for the presence of an antigen with the same epitope, or with a different but cross-reactive epitope, or for the presence of CTLs which specifically recognize the corresponding epitopes.
  • the sample will normally be a biological fluid, such as blood, urine, lymphatic fluid, amniotic fluid, semen, saliva, tears, milk, or cerebrospinal fluid, or a fraction or derivative thereof, or a biological tissue, in the form of, e.g., a tissue section or homogenate.
  • the preferred sample is blood, or a fraction or derivative thereof.
  • Assays may be divided into two basic types, hetero ⁇ geneous and homogeneous.
  • heterogeneous assays the interaction between the affinity molecule and the analyte does not affect the label, hence, to determine the amount or presence of analyte, bound label must be separated from free label.
  • homogeneous assays the interaction does affect the activity of the label, and therefore analyte levels can be deduced without the need for a separation step.
  • Assays may also be divided into competitive and non- competitive formats.
  • the competitive format the analyte competes with a labeled analyte analogue for binding to a binding partner.
  • a sandwich assay the analyte is first bound by a capture reagent, and then by a tag reagent.
  • the assay In order to detect the presence, or measure the amount, of an analyte, the assay must provide for a signal producing system (SPS) in which there is a detectable difference in the signal produced, depending on whether the analyte is present or absent (or, in a quantitative assay, on the amount of the analyte) .
  • SPS signal producing system
  • the detectable signal may be one which is visually detectable, or one detectable only with instruments.
  • Possible signals include production of colored or luminescent products, alteration of the characteristics (including amplitude or polarization) of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product.
  • a label is intended to include the discontinuance of an existing signal, or a change in the rate of change of an observable parameter, rather than a change in its absolute value.
  • the signal may be monitored manually or automatically.
  • the component of the signal producing system which is most intimately associated with the diagnostic reagent is called the "label".
  • a label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, an agglutinable particle.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 125 1, 131 I, 35 S, 14 C, and, preferably, 12S I . It is also possible to label a compound with a fluorescent compound. When the fluorescently labeled anti ⁇ body is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine .
  • fluorescence-emitting metals such as 125 Eu, or others of the lanthanide series, may be attached to the binding protein using such metal chelating groups as diethyl- enetriaminepentaacetic acid (DTPA) and ethylenediamine- tetraacetic acid (EDTA) .
  • DTPA diethyl- enetriaminepentaacetic acid
  • EDTA ethylenediamine- tetraacetic acid
  • the peptides also can be detectably labeled by coupling to a chemiluminescent compound.
  • a chemiluminescent compound The presence of the chera- iluminescently labeled antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isolumino, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bio ⁇ luminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Enzyme labels such as horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, staphylococcal nuclease, ⁇ -V-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycero phosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, /3-galactosidase, ribonuclease, glucose-6-phosphate dehydrogenase, glucoamylase and acetyl- choline esterase, are preferred.
  • the signal producing system must also include a substrate for the enzyme. If the enzymatic reaction product is not itself detectable, the SPS will include one or more additional reactants so that a detectable product appears.
  • a label may be conjugated, directly or indirectly (e.g., through a labeled antibody), covalently (e.g., with SPDP) or noncovalently, to the peptide, to produce a diagnostic reagent.
  • the peptide may be conjugated to a solid phase support to form a solid phase ("capture") diagnostic reagent.
  • Suitable supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to its target .
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • the peptides may be used as a diagnostic tool to evaluate whether other immunotherapeutic treatments (tumor vaccines of any kind, adoptive transfer of CTL, etc) are having a beneficial effect.
  • the peptides 946L (YLEPGPVTA) and 9461 (YIEPGPVTA) have low to intermediate affinity for the HLA-A2.1 molecule. This is illustrated in Figure 11. For this reason, they will be useful as control peptides for the evaluation of candidate peptide/MHC binding affinity. Because they represent a low affinity range, they can be used in laboratory studies on binding affinity of other peptides.
  • This methodology would likely include: binding the peptide to T2 cells, then evaluating lysis of the T2 cells by any of various standard methods, such as a proliferative response of the CTL, or cytokine release by the CTL exposed to the T2 cells+ peptide.
  • Fibroblasts GM126 were obtained from the National Institute of General Medical Sciences Human Genetic Mutant Cell Repository, Bethesa, MD.
  • Melanoma lines DM6, DM13, DM14, and DM93 were the gift of Drs. Hilliard F. Siegler and Timothy L. Darrow.
  • VMM1 and VMM5 are melanoma cell lines established from metastatic melanoma resected from patients at the University of Virginia (Charlottesville, VA) .
  • VBT2 squamous cell lung carcinoma
  • VAOl adenocarcinoma of the ovary
  • VAB5 adenocarcinoma of the breast
  • JY, MICH, MWF, 23.1, RPMI 1788, and Herluff are EBV-transformed B lympho- blastoid lines.
  • K562 is a NK-sensitive human erythroleukemia line.
  • the cell line T2 is derived from the fusion of a T cell line, CEM, and a human B cell mutant, LCL 721.174. This cell line expresses HLA-A2.1 molecules but has an Ag- processing defect that is associated with enhanced presenta ⁇ tion of exogenous peptides.
  • the HLA types of several cell lines are listed in Table 1.
  • HLA-A3- restricted CTL recognize shared antigens on autologous and allogeneic melanoma cells, including an HLA-A3-restricted peptide derived from Pmel-17/gpl00 and one or more peptides not yet identified, but apparently not derived from Pmel- 17/gpl00.
  • the human melanoma cell lines VMM1, VMM12, VMM18 and VMM34 were derived from patients at the University of Virginia (Charlottesville, VA) .
  • DM6 was provided by Drs. H.F. Seigler and T.L. Darrow at Duke University (Durham, NC) .
  • SkMel-2 was obtained from the
  • VMM12-EBV is an Epstein-Barr virus transformed B cell line derived from peripheral blood mononuclear cells (PBMC) of melanoma patient VMM12. Briefly, the PBMC were incubated with filtered supernatant from the
  • K562 is an NK- sensitive human erythroleukemia line.
  • T2-A3 an HLA-A3 transfectant of the antigen-processing-defective mutant human lymphoid cell line, T2 was provided by P. Cresswell.
  • HLA typing was performed by microcytotoxicity assay on autologous lymphocytes (Gentrak) . Expression of HLA-A3 by tumor cells was confirmed by staining with the monoclonal antibody (MAb) GAP-A3 provided by P. Cresswell .
  • the full-length Pmel-17 cDNA was sub-cloned from pcDNAl/neo (Invitrogen) into a modified pSCll vector adjacent to the vaccinia P7.5 early/late promoter using standard recombinant DNA methods. Standard dideoxy sequencing was used to confirm insertion and orientation.
  • a recombinant vaccinia virus expressing the protein encoded by this gene (vac-Pmel-17) was generated using published methods. Briefly, CV-1 cells were infected with the parental WR strain of vaccinia virus and transfected (Lipofectin, Gibco-BRL) with the pSCll .3-Pmel-17 plasmid.
  • Thymidine-kinase negative recombinants were amplified in 143B TK " cells in the presence of bromodeoxyuridine (Sigma, St Louis, MO) . Recombinants with beta-galactosidase activity were isolated and cloned through several rounds of plaque purification. Large-scale stocks were produced, sucrose purified, and titered in CV-1 cells. Generation of melanoma -sped fie cytotoxic T cells : CTL were generated following the detailed protocols previously reported. Malignant melanoma was resected from lymph nodes of patient VMM18.
  • Nodes were mechanically dissociated and enzy atically digested in Eagle's MEM (GIBCO, Grand Island, NY) containing 2.5% FCS, 0.1% collagenase B (Boehringer Mannheim) , 0.002% DNAase (Sigma) , penicillin 100 U/ml, streptomycin 100 ug/ml (Pen-Strept, GIBCO) at room temperature.
  • T cell lines were established from the mixture of lymphocytes and tumor obtained from the digests, using a ratio of tumor cells to lymphocytes of 1:1.
  • Goulmy personal communication
  • 5 x 10 6 irradiated (10 Gy) autologous melanoma stimulators and 10 x 10 6 irradiated (10 Gy) allogenic PBL feeders (pooled from at least three donors) .
  • the cells were cultured at 37 °C in 80mls RPMI 1640 containing 10% FCS, Pen-Strept, and 20 U/ml rIL-2 in the edge of an upright T-75 flask (Falcon) , set at a 45° angle. After five days 40ml fresh culture medium was added to the flask which was then placed upright for a further two days.
  • T lymphocytes were harvested and cryopreserved in 2 x 10 6 aliquots in 90% FCS/10% DMSO for use in cytotoxic T cell assays. This method was found to permit significant expansion of T-cell numbers without changing the specificity of the CTL line (data not shown) .
  • T cells were evaluated by flow cytometry after staining with fluorescein- or phycoerythrein-conjugated antibodies to CD3 , CD4, CD8 and CD16 (GenTrak Inc., Plymouth Meeting, PA. and Olympus Corp, Lake Success, NY) . Multiple CD8 + VMM18 CTL lines were generated following this protocol with consistent results from each. Similar methods were used for generation of CTL lines from other patients, such as VMM12.
  • Cytotoxici ty assays Cell mediated lysis of target cells was determined using a standard 4 h 51 Cr-release assay. Briefly, 5I Cr-labeled target cells were plated at 2xl0 3 cells/well in triplicate on 96-well V-bottom plates (Costar, Cambridge, MA) with indicated ratio of effector cells in a final volume of 200 microliters. Wells containing either culture medium or 1M HCl in place of the effector cells served as spontaneous and maximum 51 Cr-release controls, respectively.
  • Vaccinia infected targets were generated by infecting cells with 50 pfu/cell of appropriate recombinant vaccinia virus at 37°C for 5 h, prior to 51 Cr- labeling.
  • Antibody blocking assays were performed by incubating 5I Cr-labeled target cells with affinity purified monoclonal antibodies (MAb) for 1 h at 37°C, prior to incubation with effector CTL.
  • MAb monoclonal antibodies
  • the MAbs used included W6/32, specific for a monomorphic determinant on all human class I MHC molecules; L243, specific for a determinant on human DR molecules; and GAP-A3, specific for HLA-A3.
  • CTL were incubated with unlabeled (cold) target cells for 1 h at 37°C, prior to addition of 51 Cr-labeled (hot) targets.
  • Peptide sequences were selected from the reported human sequence of Pmel- 17/gpl00 based on predicted HLA-A3 binding motifs. These peptides were synthesized by standard Fmoc chemistry using a Gilson model AMS422 peptide synthesizer. Peptides were reconstituted in CTL assay medium (RPMI 1640, 10% FCS, antibiotics) and pre-incubated for 2 h with 2xl0 3 51 Cr labeled target cells in 100 microliters/well in 96-well plates.
  • CTL assay medium RPMI 1640, 10% FCS, antibiotics
  • Effector cells were added in 100 microliters assay medium for a final effector to target (E:T) ratio of 20:1 and the remainder of the assay was performed as in standard chromium release assays described above. Wells containing peptide and target cells but no CTL were used as controls to rule out toxicity of the peptides themselves.
  • Initial experiments were performed with unpurified synthetic peptides. Biologically active peptides identified at initial screening were then purified to >98% by reversed-phase HPLC on a Vydac C-4 column with 0.05% trifluoroacetic acid:water and an acetonitrile gradient, then re-evaluated in CTL assays.
  • HLA-A3-associated peptides were acid eluted from HLA-A3 molecules affinity-purified from melanoma cells, as previously described for A2-associated peptides-. Briefly, VMM18 melanoma cells cultured in cell factories (Nunc, Naperville, IL) , were washed three times in cold PBS, pelleted, then snap-frozen.
  • Cell pellets were detergent solubilized in 1% CHAPS, 174 mg/ml PMSF, 5 mg/ml aprotinin, 10 mg/ml leupeptin, 16 mg/ml pepstatin A, 33 mg/ml iodoacetamide, 0.2% sodium azide and 0.03 mg/ml EDTA for 1 h at 4°C. After centrifugation at 100,000 x g for 1 h at 4°C, the pellet of insoluble proteins was discarded, and the supernatant was filtered (0.2 urn), then passed over a protein A-Sepharose column precoated with MAb GAP-A3.
  • HLA-A3 restricted melanoma specific human CTL recognize one or more commonly expressed antigens
  • Cytotoxic T lymphocyte (CTL) lines were generated by repeated co-culture of lymphocytes, originally harvested from a tumor involved lymph node, with fresh or cultured autologous melanoma cells from patient VMM18 in the presence of rIL-2 as described.
  • CTL lines were derived, which lysed autologous tumor, whereas there was minimal lysis of the NK target K562, an allogeneic HLA-A3 + EBV-transformed B cell line (VMM12-EBV) or the HLA-A3 " melanoma DM6 (Fig.lA) .
  • Lysis of autologous tumor was MHC- class I restricted, based on inhibition with W6/32, a MAb specific for human class I molecules, but not L243, a MAb specific for a determinant on human DR molecules (Fig. IB) . Furthermore, inhibition observed with GAP-A3, a MAb recognizing HLA-A3, demonstrates that the VMM18 CTL recognize one or more peptides presented by HLA-A3 on the surface of the autologous melanoma cells.
  • VMM18 CTL lysed several other HLA-A3 matched allogeneic melanomas: VM 1, VMM12, DM122, and SkMel-2, indicating that one or more shared epitope (s) are presented on the surface of multiple HLA-A3 + melanomas (Table 101) .
  • lysis of allogeneic HLA-A3 matched melanoma cells by VMM18 CTL was inhibited by unlabeled (cold) autologous melanoma cells (VMM18) , but not by HLA-A3 " melanoma cells (DM6) (Fig. 2) . This confirms the existence of shared epitopes restricted by HLA-A3.
  • HLA-A3 + non-melanoma cells such as the squamous lung cancer cell line SkMes-1 and the lymphoblastoid cell line VMM12-EBV was not observed (Table 101) , indicating that these epitopes may be derived from one or more melanoma-specific proteins.
  • vac-Pmel-17 a recombinant vaccinia virus (vac-Pmel-17) expressing the full-length protein encoded by the Pmel-17 cDNA was constructed. Expression of Pmel-17 by the recombinant vaccinia was confirmed by infecting C1R-A2, an HLA-A2 + non-melanoma cell line, with vac-Pmel-17 or an irrelevant recombinant vaccinia encoding the influenza nucleoprotein, NP (vac-NP) .
  • peptides from Pmel-17/gpl00 were synthesized on the basis of peptide binding motifs for HLA-A3. These peptides were screened for their ability to sensitize allogeneic HLA-A3 + non-melanoma cells for lysis by VMM18 CTL. Two of these peptides, the nonamer ALLAVGATK and its amino terminal truncated octamer LLAVGATK, sensitized VMM12-EBV for lysis by VMM18 CTL (Table 102) .
  • the relative ability of these peptides to sensitize targets for lysis was determined in a titration assay using T2-A3, the non-melanoma HLA-A3 transfectant of the antigen processing defective mutant cell line T2.
  • Half maximal lysis was induced with 1-10 nM and > 1 uM of peptides ALLAVGATK and LLAVGATK respectively, while recognition of the HLA-A3 binding peptide QVPLRPMTYK, derived from the HIV Nef protein was not observed (Fig. 4) .
  • the nonamer peptide was able to sensitize targets for VMM18 CTL recognition at a significantly lower concentration than the octamer, suggesting that it is more likely to be the naturally processed peptide to which the CTL were primed.
  • the nonamer peptide ALLAVGATK is naturally processed and presented by melanoma cells in association with HLA-A3
  • HLA-A3 associated peptides were isolated from VMM18 melanoma cells and fractionated by reversed-phase HPLC, as described.
  • the synthetic peptide ALLAVGATK (mass of 846 and m/z of 423) was eluted under identical conditions and found in fraction 14.
  • ALLAVGATK identical to the predicted synthetic peptide. This confirms that peptide ALLAVGATK from Pmel-17/gpl00 is a naturally processed antigenic peptide, presented by HLA-A3 on melanoma cells.
  • this protein is expressed by the majority of melanoma cells and is a tissue differentiation antigen of melanocytic origin, this peptide represents a shared epitope for A3-restricted melanoma- specific CTL.
  • Analysis of HLA-A2 associated peptides eluted from the surface of melanoma cells has demonstrated that the amino acid sequences of naturally processed MHC-associated peptides may differ from their respective gene-encoded amino acid sequences because of post-translational modifications and that the gene-encoded sequence may not be presented at all.
  • HLA-A3 associated peptides from VMM18 tumor cells were evaluated directly and sequenced by tandem mass spectrometry. By this method, it has been confirmed that this peptide is naturally processed and presented by HLA-A3.
  • HLA-A2 and -A3 are two of the most commonly expressed haplotypes in Caucasian populations, representing 46% and 24% respectively.
  • the identification of an HLA-A3 restricted epitope expands the number of patients (to 60%) who might be targeted for immunization against Pmel-17 antigens. It also suggests that Pmel-17 directed immunotherapy may be an important part of immune therapy for melanoma patients of many different haplotypes.
  • VMM18 CTL Although the Pmel-17 derived peptide ALLAVGATK is recognized by VMM18 CTL, it is not recognized by CTL from another patient, VMM12. However, VMM12 CTL do recognize and lyse VMM18 melanoma cells. Because the only Class I MHC molecule shared by VMM12 and VMM18 is HLA-A3 , it is evident that at least one additional shared CTL epitope is expressed by both of these tumors.
  • Table 101 Recognition of autologous and allogeneic HLA-A3 + melanoma cell lines bv VMM 18 CTL. Targets were assayed in triplicate using an E:T ratio of 10: 1 in the three representative experiments shown.
  • GVSRQLRTK 0-8 c TL1GANASF 0 ⁇ r- ALNFPGSQK -18 r ⁇ QVWGGQPVY -6-2 ro YVWKTWGQY -22
  • VAN DEN EYNDE et al. , 1995. Anew family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J. Exp. Med. 182 (3) : 689-98.
  • KAWAKAMI et al . , 1994. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91 (14) :6458. 11. KAWAKAMI, et al . , 1994. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc. Natl. Acad. Sci. USA 91 : 3515 .
  • ADEMA et al., 1993.
  • HMB-45 are encoded by a single cDNA.
  • a recombinant vaccinia virus has been constructed that was designed to express the full-length tyrosinase protein. Appropriate expression of the tyrosinase protein was confirmed by infecting tyrosinase-negative non-melanoma cells with this newly constructed virus and demonstrating their subsequent recognition by murine tyrosinase specific T cells.
  • JY Human HLA-A2-positive lymphoblastoid cells
  • a recombinant vaccinia virus expressing the full-length tyrosinase protein, labeled, and combined with murine cytolytic T cells specific for the HLA-A2-restricted tyrosinase "D" peptide (YMDGTMSQV) , which were generated in our laboratories.
  • Recognition of the vaccinia encoded tyrosinase was ascertained by measuring target cell lysis in a standard chromium release assay. As expected, uninfected JY, and JY infected with a recombinant vaccinia encoding an irrelevant protein (NP) , were not recognized.
  • NP irrelevant protein
  • JY cells pulsed with the "D" peptide and DM6 melanoma cells served as positive controls, demonstrating the lytic potential and specificity of the T cells in this particular assay, as well as the efficacy of the vaccinia construct as a means of inducing expression of tyrosinase in a cell .
  • a panel of human cytolytic T lymphocytes (CTL) was then screened for recognition of the tyrosinase protein by the same method.
  • One human CTL line, VMM12 was found to specifically recognize tyrosinase.
  • VMM12EBV infected with recombinant vaccinia encoding tyrosinase were recognized and lysed, whereas VMM12EBV infected with a recombinant vaccinia construct encoding an irrelevant protein (NP) were not recognized, demonstrating that the recognition of VMM12EBV infected with vaccinia-tyrosinase was absolutely dependent on expression of the tyrosinase protein.
  • MHC Major Histocompatability Complex
  • VMM12EBV which express the HLA-Al, -A3, -B7, and -B14 MHC molecules
  • VMM12EBV which express the HLA-Al, -A3, -B7, and -B14 MHC molecules
  • tyrosinase-expressing recombinant vaccinia virus and VMM12 melanoma tumor cells, were recognized.
  • C1R (lymphoid) target cells that were recognized were those that expressed both HLA-Al and tyrosinase.
  • the peptide RCDICTDEY when pulsed onto HLA-A1+ non-melanoma cells (C1R-A1) , reconstitutes an epitope for VMM15 CTL. To a lesser extent, two other peptides that are longer than 9-residues, but which contain the entire KCDICTDEY sequence, also reconstitute an epitope for these CTL. None of 116 other peptides tested worked. Thus, we believe this is an epitope which can be used as an immunogen in treating or preventing melanoma in the 20-25% of patients who express HLA-Al.
  • the human melanoma cell lines VMMl, VMM12, VMM15, VMM18, VMM30 and VMM34 were derived from patients at the University of Virginia (Charlottesville, VA) .
  • Other fresh (uncultured) tumors VMM14 and VMM21 were also prepared from surgical specimens from patients at the University of Virginia.
  • DM6 was provided by Drs. H.F. Seigler and T.L. Darrow at Duke University (Durham, NC) . Immunohistochemical staining of these cell lines with S-100, HMB-45 and vimentin antibodies was characteristic of melanoma, while staining for epithelial membrane antigen and cytokeratin was negative.
  • VMM12- EBV is an Epstein-Barr virus transformed B cell line derived from peripheral blood mononuclear cells (PBMC) of melanoma patient VMM12. Briefly, the PBMC were incubated with filtered supernatant from the EBV producing cell line B-958 for 1 h at 37°C, followed by culture in RPMI 1640 media with 10% fetal calf serum (FCS) and antibiotics, plus a 1:100 dilution of PHA.
  • R562 is an NR-sensitive human erythroleukemia line.
  • T2- A3 an HLA-A3 transfectant of the antigen-processing- defective mutant human lymphoid cell line, T2 was provided by P. Cresswell. HLA typing was performed by microcytotoxicity assay on autologous lymphocytes (Gentrak) . Expression of HLA-Al by tumor cells was confirmed by staining with a monoclonal antibody (MAb) from One Lambda.
  • MAb monoclonal antibody
  • CTL l ines We have generated human melanoma-specific CTL lines by in vitro stimulation with autologous tumor, from patients whose tumors express melanocytic tissue differentiation antigens and express one or more of the MHC molecules Al, A3, B7, and B8. Methods for CTL generation have been described. (Table 111 and Figure 5) .
  • CV-1 cells were infected with the parental WR strain of vaccinia virus and transfected (Lipofectin, Gibco- BRL) with the pSCll.3-Pmel-17 plasmid.
  • Thymidine-kinase negative recombinants were amplified in 143B TR " cells in the presence of bromodeoxyuridine (Sigma) .
  • Recombinants with beta-galactosidase activity were isolated and cloned through several rounds of plaque purification. Large-scale stocks were produced, sucrose purified, and titered in CV-1 cells.
  • the resulting recombinant vaccinia viruses were used to infect the lymphoblastoid cell lines C1R-A1, C1R-A2, C1R-A3, C1R-B7, and C1R-B8, where C1R is a human lymphoblastoid line devoid of native expression of HLA-A or HLA-B region molecules, but expressing low levels of HLA-C and MHC Class II molecules.
  • C1R is a human lymphoblastoid line devoid of native expression of HLA-A or HLA-B region molecules, but expressing low levels of HLA-C and MHC Class II molecules.
  • EBV-transformed B cells with defined MHC expression were used for the infections. This resulted in transient expression of the antigens of interest. These cells were assayed for recognition by CTL in Cr51- release assays.
  • target cells were also infected with a recombinant vaccinia virus with an irrelevant DNA insert (influenza nucleoprotein, NP) .
  • NP influenza nucleoprotein
  • a negative control was uninfected C1R-MHC line and a C1R-MHC line transfected with a vaccinia construct expressing influenza nucleoprotein, vac-NP only.
  • 51 Cr-labeled target cells were plated at 1 - 2xl0 3 cells/well in triplicate on 96-well V-bottom plates (Costar, Cambridge, MA) with indicated ratio of effector cells in a final volume of 200 microliters.
  • Wells containing either culture medium or IM HCl in place of the effector cells served as spontaneous and maximum 51 Cr-release controls, respectively.
  • Vaccinia infected targets were generated by infecting cells with 50 pfu/cell of appropriate recombinant vaccinia virus at 37°C for 5 h, prior to 51 Cr-labeling.
  • Peptide synthesis and Reconsti tution wi th synthetic peptides Peptide sequences were selected from the reported human sequence of tyrosinase, based on predicted HLA-Al binding motifs (see table 10) . These peptides were synthesized by standard Fmoc chemistry using a Gilson model AMS422 peptide synthesizer. Peptides were reconstituted in CTL assay medium (RPMI 1640, 10% FCS, antibiotics) and pre-incubated for 2 h with 2xl0 3 51 Cr labeled target cells in 100 microliters/well in 96-well plates.
  • CTL assay medium RPMI 1640, 10% FCS, antibiotics
  • Effector cells were added in 100 microliters assay medium for a final effector to target (E:T) ratio of 20:1 and the remainder of the assay was performed as in standard chromium release assays described above. Wells containing peptide and target cells but no CTL were used as controls to rule out toxicity of the peptides themselves.
  • Initial experiments were performed with unpurified synthetic peptides. Biologically active peptides identified at initial screening were then purified to >98% by reversed-phase HPLC on a Vydac C-4 column with 0.05% trifluoroacetic acid:water and an acetonitrile gradient, then re-evaluated in CTL assays.
  • the CTL lines listed in Table 111 were evaluated for recognition of peptides derived from the 3 melanocytic tissue differentiation antigens listed above, in chromium-release assays, by transient infection with vaccinia constructs encoding those genes. Examples of their reactivity against HLA-matched allogeneic melanomas are shown in Figure 5. A summary of these results with vaccinia constructs are listed in Table 112 and are shown in Figure 6. Responses to tyrosinase peptides were observed in half of cases. In addition, responses to MART-1 and gplOO peptides were observed in a smaller set of CTL lines. At least two of the HLA-A1+ CTL lines recognized tyrosinase peptides in an HLA-Al-restricted manner.
  • VMM12 CTL and VMM15 CTL were assayed initially on autologous EBV-B cells as targets. Reactivity against tyrosinase was observed, so additional studies were performed to confirm the reactivity and to determine the MHC restriction (Figure 6) .
  • C1R cells that express selected Class I MHC molecules only were used as target cells.
  • C1R-A1 cells infected with vac-tyrosinase are recognized by VMM12 and VMM15 CTL, confirming that one or more tyrosinase-derived peptides are recognized by VMM12 and VMM15 CTL in association with HLA-Al.
  • the peptide representing residues 243-251 of tyrosinase reconstitutes an epitope for VMM15 CTL.
  • a set of peptides were synthesized from the defined amino acid sequence of tyrosinase, including 9-mers and longer peptides, with tyrosine (Y) at the C-terminal position and T, S, or M at position 2 and/or D, E, A, or S at position 3 ( Figure 7) . These were assayed for their ability to reconstitute epitopes for melanoma-reactive CTL VMM12 and VMM15.
  • C1R-A1 cells were pulsed with the peptide at concentrations ranging from 0.1 to 10 uM in normal assay medium (RPMI + 10% FCS) , then evaluated for recognition in a chromium-release assay.
  • RPMI + 10% FCS normal assay medium
  • three peptides were recognized by VMM15 CTL, all containing the sequence KCDICTDEY (tyrosinase residues 243-251) .
  • the peptide RCDICTDEY appears to be recognized by CTL from at least two different patients, in association with HLA-Al. Although longer peptides also are reactive, the dominant response seems to be to KCDICTDEY. This peptide is unusual in its large number of polar amino acid residues, including two aspartic acid residues, one glutamic acid residue, and two cystine residues. The tyrosine residue at position 9 and the aspartic acid at position 3 are important for binding to the MHC.
  • KCDICTDEY is associated with half-maximal lysis at approximately 1 ug/ml (1 uM) . Evaluating the possibility of increasing the potency of this activity is underway, by assessing various amino acid substitutions and their effects on CTL recognition.
  • HLA-Al-reactive HLA-Al-restricted CTL There have been two peptides described as epitopes for melanoma-reactive HLA-Al-restricted CTL. They are the MAGE-1 and MAGE-3 peptides EADPTGHSY and EVDPIGHLY While these have substantial potential value as immunogens, only a subset of melanoma patients express them. Most other MHC-associated peptide epitopes are HLA-A2 associated. However, HLA-Al is expressed in approximately 29% of patients in this country. We have previously described an HLA-A3-associated epitope from gplOO, ALLAVGATK.

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WO2001032193A1 (en) * 1999-10-29 2001-05-10 Argonex Pharmaceuticals Cytotoxic t lymphocyte-stimulating peptides for prevention, treatment, and diagnosis of melanoma
US6861234B1 (en) * 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
US20040138135A1 (en) * 2000-05-31 2004-07-15 Nicolette Charles A. Therapeutic compounds for ovarian cancer
WO2002060390A2 (en) * 2001-01-04 2002-08-08 Yale University Induction of immune responses to isoaspartyl-modified antigens
DK1453471T3 (da) 2001-11-07 2011-03-28 Mannkind Corp Ekspressionsvektorer, der koder for epitoper af antigener, og fremgangsmåde til deres konstruktion
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