WO2004045555A2 - Epitopes immunogenes pour facteur de croissance fibroplastique 5 (fgf-5) presente par hla-a3 et hla-a2 - Google Patents

Epitopes immunogenes pour facteur de croissance fibroplastique 5 (fgf-5) presente par hla-a3 et hla-a2 Download PDF

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WO2004045555A2
WO2004045555A2 PCT/US2003/037065 US0337065W WO2004045555A2 WO 2004045555 A2 WO2004045555 A2 WO 2004045555A2 US 0337065 W US0337065 W US 0337065W WO 2004045555 A2 WO2004045555 A2 WO 2004045555A2
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peptide
seq
fgf
hla
subject
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PCT/US2003/037065
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WO2004045555A3 (fr
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Ken-Ichi Hanada
James C. Yang
Donna Perry-Lalley
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Priority to CA002506684A priority Critical patent/CA2506684A1/fr
Priority to AU2003298677A priority patent/AU2003298677A1/en
Priority to EP03796431A priority patent/EP1562622A2/fr
Publication of WO2004045555A2 publication Critical patent/WO2004045555A2/fr
Publication of WO2004045555A3 publication Critical patent/WO2004045555A3/fr
Priority to US11/134,703 priority patent/US20060009393A1/en

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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This application relates to human leukocyte antigen (HLA)-A3 and HLA-A2 epitopes of fibroblast growth factor 5 (FGF-5) and uses thereof.
  • HLA human leukocyte antigen
  • FGF-5 fibroblast growth factor 5
  • Chemotherapy is largely ineffective for unresectable metastatic disease. Instead, the only U.S. Food and Drug Administration (FDA) approved therapy for metastatic disease is immunotherapy with high-dose bolus interleukin 2 (IL-2). Although IL-2 can cause regression in about 15-20% of patients, only about one third of these are complete responses. If patients attain a complete response to high-dose IL-2, their chances of being alive and free of disease after a median follow-up of over nine years is 80% (Rosenberg et al., 1998. Ann. Surg. 228:319; Fisher et al., 1997. Cancer J. Sci. Amer. 3:S70-S72).
  • FDA Food and Drug Administration
  • T-cells recognize small processed peptides from these protein antigens, which are excised and presented to T-cells in a surface cleft of a specific MHC molecule (Braciale. 1992. Curr. Opin. Immunol. 4:59-62).
  • processed epitopes that bind to Class I MHC are typically 8-10 a ino acids in length and conform to certain structural motifs for each MHC molecule.
  • Studies using the exact epitopes recognized within the melanoma-associated antigens have revealed that there are many mechanisms used to generate immunogenic epitopes, such as out of frame translation from alternative ORFs (Wang et al, 1996. J. Exp. Med.
  • Augmented T-cell responses may improve IL-2 therapy.
  • administration of melanoma-reactive T-cells along with IL-2 produced a response rate higher than that of IL-2 alone, and responses were observed in patients who had previously not responded to IL-2 alone (Rosenberg et al, 1994. J. Natl. Cancer Inst. 86:1159-66).
  • a peptide vaccine derived from the melanoma/melanosomal antigen, GP100 when given with high-dose IL-2 resulted in a response rate over 30% (Rosenberg et al, 1998. Nat. Med. 4:321-7).
  • RCC antigens that contain naturally- processed peptide epitopes, and which are highly expressed on a significant proportion of RCC tumors but with little or no expression on normal adult tissues.
  • tumor antigens which extend effective cancer immunotherapy beyond just melanoma and RCC, for example to common adenocarcinomas (such as breast, prostate, and pancreatic cancer) which constitute a large portion of all human cancers and for which there are few or no successful immunotherapy approaches.
  • HLA-A3 and HLA-A2 FGF-5 epitopes which can stimulate an immune response, and in some examples, can be used to treat an FGF-5 expressing (or overexpressing) tumor.
  • HLA-A molecules are Class I MHC antigens, which serve as target antigens for immune recognition and killing.
  • HLA-A2 and HLA- A3 molecules are alternate forms of HLA-A molecules, which can have small variations in their nucleotide sequences, which results in cell surface glycoproteins with small variations, a change of one or more amino acids, and/or a change in three-dimensional structure.
  • HLA-A2 and HLA-A3 molecules can recognize distinct epitopes of a single protein, such as FGF-5.
  • FGF-5 HLA-A3 epitopes are disclosed herein.
  • the disclosure provides purified, imniunogenic peptides which include SEQ ID NO: 17, as well as variants thereof that can stimulate an immune response, such as SEQ ID NO: 9.
  • such variants are recognized by, or can generate an immune cell (nominally a T-cell) that specifically reacts with SEQ ID NO: 9.
  • FGF-5 HLA-A3 epitopes include, but are not limited to, a sequence which includes the peptide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, as well as variants thereof that can stimulate an immune response.
  • An example of a particular FGF-5 HLA-A2 epitope includes, but is not limited to a peptide sequence which includes the peptide sequence shown in SEQ ID NO: 7, as well as variants thereof that can stimulate an immune response.
  • such variants are recognized by, or can promote production of an immune cell (nominally a T cell), that specifically reacts with SEQ ID NO: 7.
  • Nucleic acids encoding the disclosed peptides are also encompassed by this disclosure, as well as host cells expressing the peptides.
  • Methods are also disclosed for treating an FGF-5 expressing or overexpressing tumor, such as an adenocarcinoma, for example RCC, using the disclosed HLA- A3 and HLA-A2 FGF-5 epitopes, or a nucleic acid encoding such peptides.
  • Subjects having an FGF-5 expressing or overexpressing tumor can receive the disclosed peptides alone, or in the presence of other therapeutically effective molecules, such as IL-2.
  • Administration of the disclosed FGF-5 HLA-A2 and HLA- A3 epitoptic peptides (or nucleic acids encoding them) can be used to induce an immune response in a subject against these clinically-relevant tumor antigens.
  • the HLA haplotype of the subject is determined prior to adniinistration of the disclosed peptides and nucleic acids.
  • Methods of producing antibodies specific for an FGF-5 antigen are also disclosed, for example by introducing the disclosed FGF-5 HLA-A2 and HLA- A3 antigens (or variants thereof) into a subject, and allowing the subject to generate antibodies that recognize such antigens, or the variants thereof.
  • FIG. 1 is a graphical representation showing FGF-5 sequences that were (open bars) or were not (filled bars) recognized by a renal cancer-reactive T-cell clone, Clone 2.
  • FIG. 2 shows the sequences of the FGF-5 internal deletion mutants. All constructs were designed to start with the Kozak consensus sequence and end with the termination codon TAA. Numbers in brackets stand for the position of the first and the last amino acids. Numbers following ⁇ indicate internally deleted amino acid residues.
  • the degree of CTL recognition is categorized as follows depending on IFN- ⁇ secretion: (++) >1000 pg/ml, (+) 200-1000 pg/ml, (+/-) 100-200 pg/ml, (-) ⁇ 100 pg/ml.
  • FIG. 3 is bar graph showing the results of alanine substitutions, to determine which FGF-5 residues play a role in Clone 2 recognition.
  • FIG. 4 is a bar graph showing FGF-5 peptides that generate a significant IFN- ⁇ response.
  • FIG. 5 is a graph showing the titration of three FGF-5 peptides.
  • the fusion peptides (NTYASPRFK, NTYASLPRFK, NTYFLPRFK; SEQ ID NOS: 1 ,2 and 10, respectively) were pulsed onto the autologous EBV-B cell line at the concentrations indicated and their recognition by C2 was assessed by IFN- ⁇ assay. Error bars represent the standard deviation of duplicate IFN- ⁇ determinations.
  • FIG. 6 is a schematic representation of plasmids with termination codons inserted between the two dete ⁇ nant-encoding fragments of G8.
  • FIG.7 is a bar graph showing that determinant generation of the HLA- A3 epitope is not the result of ribosome skipping or RNA splicing. Error bars represent the standard deviation of the duplicate IFN- ⁇ determination.
  • FIG. 8A is a schematic representation of extended FGF-5 synthetic peptides.
  • FGF-5 (173-220; SEQ ID NO: 11).
  • the 48-mer peptide lacks the first N-terminal asparagine of NTYAS but includes C-terminal PRFK; FGF-5 (161-212; SEQ ID NO: 12).
  • the 52-mer peptide includes NTYAS but lacks PRFK; FGF-5 (172-220 SEQ ID NO: 13).
  • the 49-mer peptide starts with NTYAS and ends with PRFK; FGF-5 (161- 220; SEQ LD NO: 14).
  • the 60-mer peptide is encoded by the G8 plasmid in FIG. 1 (SEQ ID NO: 15).
  • FIGS. 8B and 8C are bar graphs showing the recognition of the peptides in FIG. 8A by a control RCC-reactive CTL from the same patient not recognizing FGF-5 (B) or FGF-5 -reactive Clone 2 CTL (C). Functional specificity of each CTL line is shown in each side bar using the autologous EBV-B cell line and the autologous RCC cell line as targets.
  • FIG. 9 is a graph showing that fresh and fixed EBV-B cells have a similar capacity to present the 9-mer determinant NTYASPRFK (SEQ LD NO: 1). Error bars represent the standard deviation of duplicate IFN- ⁇ determination.
  • FIGS. 10A-10D are graphs showing the ability of HPLC fractionated (A) synthetic 9-mer (SEQ ID NO: 1), (B) synthetic 49-mer (SEQ ID NO: 13), and acid- stripped peptides from (C) COS-A3 or (D) COS-A3/FGF-5 to be recognized by Clone 2. Error bars represent the standard deviation of duplicate IFN- ⁇ determination.
  • FIGS. 11A and 11B are bar graphs showing that presentation of both MHC class I-restricted peptides was blocked by clasto-Lactacystin ⁇ -lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport.
  • Open bars untreated cells
  • filled bars cells incubated with clasto-Lactacystin ⁇ -lactone.
  • RCC cell line was infected with either an adenovirus encoding GFP (open bars) or TAP-1 inhibitor ICP47 (filled bars). Bars represent the average IFN- ⁇ secretion and the error bars, the standard deviation of duplicate samples. Similar results were repeated and representative results are shown.
  • FIG. 12 is a bar graph showing CTL clones that recognized an FGF-5 HLA-A2 epitope.
  • FIG. 13 is a bar graph showing the results of a LI 18F amino acid substitution (A516C nucleotide substitution) in an FGF-5 HLA-A2 epitope.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 shows an amino acid sequence of a 9-mer FGF-5 HLA-A3 epitope.
  • SEQ ID NO: 2 shows an amino acid sequence of a 10-mer FGF-5 HLA- A3 epitope.
  • SEQ ID NOS: 3-6 show amino acid sequences, which are variants of SEQ ID NO: 1, which function as an FGF-5 HLA- A3 epitope.
  • SEQ ID NO: 7 shows an amino acid sequence of a 10-mer FGF-5 HLA-A2 epitope.
  • SEQ ID NO: 8 shows an amino acid sequence variant of SEQ ID NO: 7 (L118F), which does not function as an FGF-5 HLA-A2 epitope.
  • SEQ ID NO: 9 shows amino acids 3 to 8 (YASPRF) of SEQ ID NO: 1; that is amino acids 174-176 and 217-219 of FGF-5.
  • SEQ ID NO: 10 shows a variant amino acid sequence of SEQ ID NO: 1, which does not function as an FGF-5 HLA- A3 epitope.
  • SEQ ID NO: 11 shows amino acids 173-220 of FGF-5.
  • SEQ ID NO: 12 shows amino acids 161-212 of FGF-5.
  • SEQ ID NO: 13 shows amino acids 172-220 of FGF-5.
  • SEQ ID NO: 14 shows amino acids 161-220 of FGF-5, the 60-mer peptide encoded by the G8 plasmid in FIG. 1.
  • SEQ ID NO: 15 shows an FGF-5 nucleic acid sequence; Genbank Accession No: M37825.
  • SEQ ID NO: 16 shows an FGF-5 protein sequence encoded by SEQ ID NO: 15;
  • SEQ ID NO: 17 shows a variant FGF-5 HLA-A3 eptiope.
  • SEQ ID NO: 1 or SEQ ID NO: 3 refers to SEQ ID NO: 1, SEQ ID NO: 3, or a combination of both SEQ ID NO: 1 and SEQ ID NO: 3.
  • “comprises” means “includes.”
  • “comprising an FGF-5 HLA-A3 epitope and IL-2” means “including an FGF-5 HLA- A3 epitope and 11-2,” without excluding additional elements.
  • Agent Any substance, including, but not limited to, an antibody, chemical compound, molecule, peptidomimetic, or protein.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are adrninistered, such as injected or absorbed, to an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • the term "antigen" includes all related antigenic epitopes.
  • Antineoplastic agent A drug or biologic that decreases or in some examples inhibits the proliferation of neoplastic cells. In one example, administration of an antineoplasitic agent to neoplastic cells arrests their growth or causes regression of a tumor.
  • antineoplasitic agents include, but are not limited to, alkylating agents (such a vincristine, vinblastine or taxol), anthracycline antibiotics such as daunorubicin and doxorubicin, hormonal therapies such as tamoxifen, and miscellaneous agents such as cis-ch ' amniinedicHoroplatimun (II), and hydroxyurea.
  • Antineoplastic agents also include biologies, such as IL-2 and alpha-interferon, and immunotherapy, for example with bacille Calmette-Guerin (BCG). Protocols for administration of such agents are known in the art, and examples can be found in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 17 th edition, section XIII.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Conservative substitution One or more amino acid substitutions for amino acid residues having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, a conservative substitution is an amino acid substitution in an antigenic epitope of an FGF-5 peptide that does not substantially affect the ability of an FGF-5 reactive T-cell to recognize the peptide.
  • a conservative substitution is an amino acid substitution in an antigenic epitope of an FGF-5 peptide, such as a conservative substitution in any of SEQ ID NOS: 1-7, 9 and 17, which does not significantly decrease recognition of the epitope by Clone 2 (for HLA- A3 FGF-5 epitopes, such as SEQ ID NO: 1-6, 9 and 17) or significantly decrease recognition of the epitope by an HLA-A2 positive tumor cell (for HLA-A2 FGF-5 epitopes, such as SEQ ID NO: 7).
  • Methods that can be used to determine the amount of recognition by a variant epitope are disclosed herein (for example, see Examples 1-3).
  • an alanine scan can be used to identify which amino acid residues in an HLA-A2 or -A3 FGF-5 epitope can tolerate an amino acid substitution.
  • recognition is not decreased by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid (such as those listed below), is substituted for one or more native amino acids.
  • one conservative substitution is included in the peptide, such as a single conservative amino acid substitution in any of SEQ ID NOS: 1-7, 9 or 17.
  • two conservative substitutions are included in the peptide.
  • three conservative substitutions are included in the peptide.
  • a polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR.
  • a polypeptide can be produced to contain one or more conservative substitutions by using standard peptide synthesis methods.
  • Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place.
  • amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; He or Val for Leu; Arg or Gin for Lys; Leu or lie for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and He or Leu for Val.
  • Deletion The removal of a sequence of a nucleic acid, for example DNA, the regions on either side being joined together.
  • Degenerate variant A polynucleotide encoding an FGF-5 epitope polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the FGF-5 polypeptide encoded by the nucleotide sequence is unchanged.
  • Enhance To improve the quality, amount, or strength of something. In one example, a therapy enhances the immune system if the immune system is more effective at reducing tumors than in the absence of the therapy.
  • an FGF- 5 HLA-A2 or HLA- A3 epitope, or combinations thereof enhances the benefit of IL-2 therapy in a subject having one or more tumors.
  • Such enhancement can be measured using any bioassay known in the art, for example, an ELISA assay or microscopy.
  • an FGF-5 HLA-A2 or HLA-A3 epitope, or combinations thereof enhances an immune response as measured by clinical response.
  • a clinical response is an increase in a population of immune cells, such as an increase of at least about 10%, for example at least 20 or even at least 50%, compared to the number of immune cells in the absence of the therapy.
  • Another example of a clinical response is a measurable reduction in the size of a tumor, for example a reduction in size of at least 5%, such as at least about 10%, at least 20, or even at least 50%.
  • Epitope An antigenic determinant. Chemical groups or peptide sequences on a molecule that are antigenic, that is, those that elicit a specific immune response. An antibody binds a particular antigenic epitope, or a T-cell reacts with a particular antigenic epitope bound to a specific MHC molecule.
  • FGF-5 HLA-A2 and HLA- A3 epitopes are peptide sequences that recognize distinct regions of FGF-5, but both can elicit an immune response against FGF-5.
  • Exemplary FGF-5 HLA-A2 epitopes include, but are not limited to, SEQ ID NO: 7 as well as variants, fragments, and fusions thereof that retain the ability to stimulate an immune response against FGF-
  • Exemplary FGF-5 HLA-A3 epitopes include, but are not limited to, SEQ ID NOS: 1-
  • Fibroblast growth factor 5 Includes both naturally occurring and recombinant FGF-5 cDNA, RNA, or protein from any organism, as well as FGF-5 fragments and FGF-5 variants that retain full or partial FGF-5 biological activity.
  • Exemplary FGF-5 gene sequences include Genbank Accession Nos: M37825 (human) and NM_010203 (mouse) and exemplary amino acid sequences include Genbank Accession Nos: AAB06463 (human) andNP_034333 (mouse).
  • FGF-5 sequences are publicly available for several other organisms.
  • FGF-5 is over-expressed in a variety of tumors, such as adenocarcinomas, such as cancers of the breast, kidney (renal cell carcinoma), prostate, bladder, and pancreas.
  • adenocarcinomas such as cancers of the breast, kidney (renal cell carcinoma), prostate, bladder, and pancreas.
  • FGF-5 Expressing Tumor A tumor which expresses or over-expresses wild- type or mutant FGF-5. Examples of such tumors include, but are not limited to: a carcinoma, for example an adenocarcinoma, such as cancers of the breast, kidney (renal cell carcinoma), prostate, bladder, and pancreas.
  • sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, insertions, and combinations thereof.
  • an FGF-5 eptiope including one or more sequence alterations retains a function of the unaltered epitope.
  • the variant FGF- 5 eptiope specifically binds an antibody that also binds to an unaltered form of an FGF- 5 eptiope.
  • the variant FGF-5 eptiope retains the ability to be recognized by Clone 2 (for HLA-A3 FGF-5 epitopes, such as SEQ ID NO: 1, 3, 4, 5 or 6) or by an HLA-A2 positive tumor cell (for HLA-A2 FGF-5 epitopes, such as SEQ ID NO: 7).
  • a particular peptide binds an antibody
  • a functional equivalent of that particular peptide is another peptide that binds the same antibody.
  • a functional equivalent includes peptides which have the same binding specificity as a polypeptide, and which may be used as a reagent in place of the polypeptide (such as in a diagnostic assay or vaccine).
  • a functional equivalent includes a polypeptide having a discontinuous binding sequence, and the antibody binds a linear epitope.
  • This polypeptide is functionally equivalent to SEQ ID NO: 7 if the three dimensional structure of the polypeptide is such that it can bind a monoclonal antibody that binds SEQ ID NO: 7, andor if it retains the ability to be recognized by an HLA-A2 positive tumor cell (see Example 3).
  • Haplotype A set of alleles of a group of closely linked genes, such as the human leukocyte antigen (HLA) complex, which are usually inherited as a unit, an individual inheriting a complete haplotype from each parent. In one example, it is the genetic constitution of an individual at a set of linked genes.
  • HLA human leukocyte antigen
  • Haplotyping or tissue typing A method used to identify the haplotype or tissue types of a subject, for example by determining which HLA locus (or loci) is expressed on the lymphocytes of a particular subject.
  • the HLA genes are located in the major histocompatibility complex (MHC), a region on the short arm of chromosome 6, and are involved in cell-cell interaction, immune response, organ transplantation, development of cancer, and susceptibility to disease.
  • MHC major histocompatibility complex
  • the most widely used method for haplotyping uses the polymerase chain reaction (PCR) to compare the DNA of the person, with known segments of the genes encoding MHC antigens. The variability of these regions of the genes determines the tissue type or haplotype of the subject.
  • Serologic methods can be used to detect serologically defined antigens on the surfaces of cells. HLA-A, -B, and -C determinants can be measured by known serologic techniques. Briefly, lymphocytes from the subject (isolated from fresh peripheral blood) are incubated with antisera that recognize all known HLA antigens. The cells are spread in a tray with microscopic wells containing various kinds of antisera.
  • the cells are incubated for 30 rninutes, followed by an additional 60-minute complement incubation. If the lymphocytes have on their surfaces antigens recognized by the antibodies in the antiserum, the lymphocytes are lysed. A dye can be added to show changes in the permeability of the cell membrane and cell death. The proportion of cells destroyed by lysis indicates the degree of histologic incompatibility. If, for example, the lymphocytes from a person being tested for HLA- A3 are destroyed in a well containing antisera for HLA-A3, the test is positive for this antigen group.
  • Immune response A change in immunity, for example, a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an "antigen-specific response").
  • an immune response is a T cell response, such as a CD4 + response or a CD8 + response.
  • the response is a B cell response, and results in the production of specific antibodies.
  • an increased or enhanced immune response is an increase in the ability of a subject to fight off a disease, such as a viral infection or tumor.
  • Immune stimulatory composition A pharmaceutical composition which includes one or more FGF-5 HLA-A2 eptitope antigens, HLA- A3 eptitope antigens, or combinations thereof, which when administered to a subject, results in the subject producing antibodies against the antigen(s).
  • the subject's response results in treatment of the subject suffering from an FGF-5 expressing or over-expressing tumor.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids and proteins which have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.
  • Leukocyte Cells in the blood, also termed “white cells,” that are involved in defending a subject against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 main types, subdivided between 2 main groups: polymo homnuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes). Generally, when a subject has an infection, the production of leukocytes increases.
  • Lymphocytes A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B-cell and T-cells.
  • Malignant Cells which have the properties of anaplasia invasion and metastasis.
  • Modulating an Immune Response Includes the ability to increase or decrease an immune response in a subject, such as the ability to stimulate a CTL immune response, such as an HLA- A3- or HLA-A2-restricted CTL response, against FGF-5 expressing or over-expressing tumors, by a desired amount, for example by at least 10% as compared to a response in the absence of an HLA- A3 peptide, HLA-A2 peptide, or combinations thereof.
  • Agents that modulate an immune response include, but are not limited to: FGF-5 polypeptides (including fragments, variants, fusion proteins, and polymorphisms thereof, such as SEQ ID NOS: 1-7, 9 and 17), FGF-5 nucleic acid molecules encoding FGF-5- polpeptides, FGF-5 specific binding agents, FGF-5 antisense molecules, and immunoreactive sensitized T cells sensitized with FGF-5.
  • FGF-5 polypeptides including fragments, variants, fusion proteins, and polymorphisms thereof, such as SEQ ID NOS: 1-7, 9 and 17
  • FGF-5 nucleic acid molecules encoding FGF-5- polpeptides include FGF-5 specific binding agents, FGF-5 antisense molecules, and immunoreactive sensitized T cells sensitized with FGF-5.
  • Neoplasm Abnormal growth of cells.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Oligonucleotide A linear polynucleotide (such as DNA or RNA) sequence of at least 9 nucleotides, for example at least 15, 18, 24, 25, 27, 30, 50, 100 or even 200 nucleotides long.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Peptide A chain of amino acids of which is at least 4 amino acids in length, regardless of post-translational modification (such as glycosylation or phosphorylation).
  • a peptide is at least 6 amino acids in length, such as at least 8, 9, 10, 11, or 12 amino acids in length.
  • a peptide is about 4 to about 30 amino acids in length, for example about 8 to about 25 amino acids in length, such as from about 9 to about 15 amino acids in length, for example about 9-10 amino acids in length.
  • a peptide is an FGF-5 epitope, such as a sequence that includes any of SEQ ID NOS: 1-7, 9 and 17 (or variants, fragments, or fusions thereof).
  • Pharmaceutical agent or drug A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • Polynucleotide A linear nucleic acid sequence of any length. Therefore, a polynucleotide includes molecules which are at least about 15, 24, 27, 30, 50, 100, 200, 500, 1000, or 5000 nucleotides in length, and also nucleotides as long as a full length cDNA.
  • An FGF-5 polynucleotide encodes an FGF-5 peptide.
  • Preventing or treating a disease refers to inhibiting the full development of a disease, for example preventing development or metastasis of a tumor in a person having an FGF-5 expressing tumor.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the presence of an FGF-5 expressing tumor, such as halting the progression of a tumor, reducing the size of the tumor, or even elimination of the tumor.
  • Promoter An array of nucleic acid control sequences that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (Bitter et al, Meth. Enzymol 153:516-44, 1987).
  • promoters include promoters derived from the genome of mammalian cells (such as a metallothionein promoter) or from mammalian viruses (such as the retrovirus long terrriinal repeat; the adenovirus late promoter; or the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used. A polynucleotide encoding an FGF-5 epitope (or variant, fragment or fusion thereof) can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that may accompany it.
  • a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.
  • an FGF-5 peptide is purified when at least 60% by weight of a sample is composed of the peptide, for example when 75%, 95%, or 99% or more of a sample is composed of the peptide.
  • methods that can be used to purify an antigen include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 17). Protein purity can be determined by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high-pressure liquid chromatography; sequencing; or other conventional methods.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques.
  • a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
  • an FGF-5 specific binding agent is an agent that binds substantially to an FGF-5 polypeptide.
  • the specific binding agent is a monoclonal or polyclonal antibody.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of an FGF-5 peptide, disclosed herein, will possess a relatively high degree of sequence identity when aligned using standard methods.
  • Biol. 215:403-10, 1990 is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • NCBI National Center for Biotechnology Information
  • tblastn sequence analysis programs
  • Variants of an FGF-5 peptide are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of FGF-5 using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 90%, at least 95%, at least 98%, or even at least 99% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85%, at least 90%, at least 95%, or 98% depending on their similarity to the reference sequence. Methods for deterrnining sequence identity over such short windows are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Maryland.
  • T Cell A white blood cell critical to the immune response.
  • T cells include, but are not limited to, CD4 + T cells and CD8 + T cells.
  • a CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8 + T cells carry the "cluster of differentiation 8" (CD8) marker.
  • a CD8 T cells is a cytotoxic T lymphocytes.
  • a CD8 cell is a suppressor T cell.
  • Therapeutically active molecule An agent, such as an FGF-5 epitope, for example SEQ ID NO: 1-7, 9 or 17 (or variants, fragments, or fusions thereof), that can induce an immune response, as measured by clinical response (for example increase in a population of immune cells, or measurable reduction in the size of a tumor).
  • Therapeutically active molecules can also be made from nucleic acids. Examples of nucleic acid based therapeutically active molecules are a nucleic acid sequence that encodes an FGF-5 epitope, wherein the nucleic acid sequence is operably linked to a control element such as a promoter.
  • Therapeutically active agents can also include organic or other chemical compounds that mimic the effects of the peptide.
  • Therapeutically Effective Amount The preparations disclosed herein are administered in therapeutically effective amounts.
  • An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. Further information about determination of a therapeutically effective dose is provided in Example 12.
  • a virus or vector "transduces” or “transfects” a cell when it transfers nucleic acid into the cell.
  • a cell is “transformed” by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transgene An exogenous nucleic acid sequence supplied by a vector.
  • a transgene encodes an FGF-5 polypeptide, such as an FGF-5 HLA- A3 or HLA-A2 epitope.
  • variants or fragments or fusion proteins include variants, fragments, and fusions thereof.
  • DNA sequences which encode for an epitope or fusion thereof, or a fragment or variant of thereof can be engineered to allow the protein to be expressed in eukaryotic cells or organisms or bacteria.
  • the DNA sequence can be altered and operably linked to other regulatory sequences.
  • the final product, which contains the regulatory sequences and the therapeutic protein, is referred to as a vector.
  • This vector can be introduced into eukaryotic or prokaryotic cells. Once inside the cell the vector allows the protein to be produced.
  • a fusion antigen comprising an FGF-5 HLA- A3 or HLA-A2 epitope (or variants, polymorphisms, mutants, or fragments thereof) linked to other amino acid sequences that do not inhibit the desired activity of the protein, for example the ability to stimulate an immune response.
  • the other amino acid sequences are at least 8, 9, 10, 12, 15, 20, 30, or 50 amino acid residues in length.
  • the DNA can be altered in numerous ways without affecting the biological activity of the encoded protein. For example, PCR can be used to produce variations in the DNA sequence which encodes an antigen.
  • Such variants can be variants optimized for codon preference in a host cell used to express the protein, or other sequence changes that facilitate expression.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art.
  • a vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell.
  • a vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • a vector is a viral vector.
  • Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
  • a renal cancer-reactive T-cell clone Clone 2
  • TIL tumor-infiltrating lymphocytes
  • This clone was HLA- A3 restricted and recognized autologous tumor as well as a number of allogeneic RCC lines also expressing HLA- A3.
  • Expression cloning of the antigen recognized by Clone 2 demonstrated that the RCC-associated antigen being recognized was unmutated fibroblast growth factor 5 (FGF-5) (Hanada et al, 2001. Cancer Res. 61:5511-6).
  • FGF-5 is expressed by a variety of tumors including bladder carcinoma and pancreatic adenocarcinoma (Yoshimura et al, 1996.
  • FGF-5 has paracrine, and possibly autocrine, trophic activity on some cultured human cancer lines and has been described to be angiogenic in vivo in a porcine model (Giordano et al, 1996. Nat. Med. 2:534-9). Many of these activities have the potential of contributing to the malignant phenotype of some cancers and make FGF-5 an attractive target for immunotherapy.
  • FGF-5 HLA-A2 and HLA-A3 epitopes are provided.
  • substantially purified FGF-5 HLA-A2 and HLA-A3 epitopes are provided, such as SEQ ID NOS: 1-7, 9, and 17, as well as variants, fusions, and fragments thereof that can elicit or stimulate an immune response, or both.
  • an FGF-5 HLA- A3 epitope is at least eight amino acids long, and includes the sequence Tyr-Ala- (A 3 )-(A 4 )-Arg-Phe wherein A 3 is Ala or Ser and A 4 is Ala or Pro (SEQ ID NO: 17).
  • the disclosed FGF-5 HLA-A2 and HLA- A3 epitopes, and methods of using them may comprise, consist, or consist essentially of any of the disclosed sequences shown in any of SEQ ID NOS: 1-7, 9, and 17 or variants or fusions thereof that retain the anti-FGF-5 immunogenic activity of the original sequences.
  • a T-cell that recognizes an FGF-5 HLA-A2 or HLA- A3 epitope can also recognize a variant of the FGF-5 HLA-A2 or HLA- A3 epitope of interest.
  • such variant immunogenic peptide sequences can stimulate propagation of an immune cell (such as a T-cell).
  • the disclosed peptides are immunogenic, and can be used to elicit an immune response against an FGF-5 expressing tumor in a subject, such as those subjects having an HLA-A2 allele, an HLA-A3 allele, or both.
  • One skilled in the art, given the present disclosure can purify the disclosed peptides using standard techniques for protein purification.
  • substantially pure polypeptides will yield a single major band on a non-reducing polyacrylamide gel.
  • the purity of a polypeptide can also be determined by amino- terminal amino acid sequence analysis. Minor modifications of the primary amino acid sequence of the disclosed FGF-5
  • HLA-A2 and HLA- A3 epitopes can result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein.
  • modifications can be deliberate, as by site-directed mutagenesis, or spontaneous. All of the polypeptides produced by these modifications are included herein as long as an activity of the variant peptide, such as the ability to induce an immune response or the ability to recognize the appropriate HLA-A2 or HLA-A3 expressing cell, still exists.
  • such variants have at least 80% identity to the disclosed sequences. Particular examples of variants include those having one or more conservative amino acid substitutions.
  • a variant includes a single amino acid substitution, such as a single conservative amino acid substitution in any of SEQ ID NOS: 1, 3-7, 9 and 17.
  • a variant includes two amino acid substitutions, or three amino acid substitutions, for example in any of SEQ ID NOS: 1, 3-7, 9 and 17.
  • the FGF-5 HLA-A2 and HLA- A3 epitopes disclosed herein can be at least 6 amino acids in length, such as at least 8, 9, 10, 11, 12, 15, 20 or even 30 amino acids in length.
  • fusion proteins including an FGF-5 HLA-A2 or HLA- A3 epitope can be even longer.
  • the FGF-5 HLA-A2 and HLA-A3 epitopes disclosed herein can be no more than about 250 amino acids, such as no more than about 100, 75, 50, 40, 30, 20 or even 15 amino acids.
  • Polynucleotides encoding the disclosed peptides are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the peptide. It is understood that all polynucleotides encoding an FGF-5 HLA-A2 epitope, HLA- A3 epitope, or both, are also included herein, as long as they encode a polypeptide with the recognized activity, such as the ability to induce an immune response or the ability to recognize the appropriate HLA-A2 or HLA-A3 expressing cell.
  • the disclosed polynucleotides include sequences that are degenerate as a result of the genetic code, as long as the amino acid sequence the FGF-5 HLA-A2 or HLA- A3 epitope encoded by the nucleotide sequence is functionally unchanged.
  • the polynucleotides encoding an FGF-5 HLA-A2 or HLA- A3 epitope can include a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single and double forms of DNA.
  • fragments of the above-described nucleic acid sequences that are at least 15 bases in length, such as at least 27 bases, such as at least 30 bases, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the disclosed FGF-5 HLA-A2 or HLA- A3 epitope (such as a polynucleotide that encodes any one of SEQ ED NOS: 1-7, 9, and 17, or nucleotide sequences that include such sequences) under physiological conditions.
  • nucleotide sequences encoding the disclosed peptides include the disclosed sequences, degenerate sequences, and sequences that encode conservative variations thereof.
  • DNA sequences encoding a disclosed FGF-5 HLA-A2 or HLA- A3 epitope can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell can be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known.
  • FGF-5 HLA-A2 or HLA-A3 epitope polynucleotide sequences can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that has been manipulated by insertion or incorporation of the FGF-5 HLA-A2 or HLA- A3 epitope genetic sequences.
  • Polynucleotide sequences which encode an epitope can be operatively linked to expression control sequences. "Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (such as ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter. Promoters include a niinimal sequence sufficient to direct transcription.
  • Promoter-dependent gene expression can be used to control cell-type specific expression, tissue-specific expression, or inducible by external signals or agents expression. Promoters can be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, can be used (Bitter et al, Meth. Enzymol 153:516-44, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
  • promoters derived from the genome of mammalian cells such as the metallothionein promoter
  • mammalian viruses such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences of the disclosure.
  • the polynucleotide encoding an FGF-5 HLA-A2 epitope, HLA-A3 epitope, or both, can be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present disclosure include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al, 1987, Gene 56:125), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (such as T7, metallothionein I, or polyhedron promoters).
  • a promoter such as T7, metallothionein I, or polyhedron promoters.
  • Polynucleotide sequences encoding an FGF-5 HLA-A2 or HLA-A3 epitope can be expressed in prokaryotes or eukaryotes.
  • Hosts include, but are not limited to, microbial, yeast, insect and mammalian cells and organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences encoding an FGF-5 HLA-A2 or HLA-A3 epitope.
  • Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding an FGF-5 HLA-A2 or HLA-A3 epitope, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • the method includes administering to the subject a therapeutically effective amount of one or more FGF-5 HLA-A2 epitopes, HLA- A3 epitopes (or nucleic acid encoding such an epitope), or both, thereby treating the tumor, for example by halting progression of the tumor, by causing regression of the tumor, or retarding growth of the tumor.
  • the HLA haplotype of the subject such as a human subject, is determined prior to administering a therapeutically effective amount of FGF-5 HLA-A2 epitope, HLA- A3 epitope, or combinations thereof.
  • FGF- 5 expressing or over-expressing tumors include, but are not limited to, adenocarcinomas, such as RCC or adenocarcinomas of the breast, prostate, and bladder.
  • the method includes administering to the subject a therapeutically effective amount of one or more FGF-5 HLA-A2 or HLA- A3 epitopes (or nucleic acid molecule encoding such an epitope), or combinations thereof, thereby eliciting an immune response in a subject.
  • an immune response are a B cell or a T cell response.
  • the HLA haplotype of the subject is determined prior to administering a therapeutically effective amount of FGF-5 HLA-A2 or HLA- A3 epitope. For example, if the subject is determined to have at least one HLA-A2 allele, the subject can be administered an FGF-5 HLA-A2 epitope.
  • Methods of generating antibodies specific for an FGF-5 antigen are disclosed.
  • the method includes administering to a subject a disclosed FGF-5 HLA-A2 or HLA- A3 antigen.
  • the subject is an experimental animal, such as a mouse or rabbit.
  • the subject is a human.
  • one or more FGF-5 HLA-A2 or HLA- A3 epitopes is utilized.
  • FGF-5 HLA-A2 and HLA- A3 epitope variants, fragments, and fusions can be employed in the pharmaceutical compositions, and can include one or more amino acid additions, amino acid deletions, amino acid replacements, or by isostereomer (a modified amino acid that bears close structural and spatial similarity to the original amino acid) substitutions, and isostereomer additions, so long as the sequences are recognized by, or can generate, and immune cell.
  • a variant of SEQ ID NO: 7 will be recognized by an immune cell that recognizes SEQ ID NO: 7.
  • such variants, fragments, and fusions provide an advantage, such as increasing the solubility or immieuxicity of the eptitope, or easing linking or coupling of the epitope.
  • the peptides included in the pharmaceutical composition can form neutralizing antibodies to an FGF-5 HLA-A2 or HLA- A3 epitope.
  • the disclosed FGF-5 HLA-A2 and HLA-A3 epitopes can also be engineered to include other amino acids (to generate a fusion protein), such as residues of various moieties, such as additional amino acid segments or polysaccharides. Examples include, but are not limited to, moieties which augment or induce antigen processing, epitope stability or manufacture, or delivery within the body to sites appropriate for immunization or recognition by immune cells.
  • an amino acid chain corresponding to an additional antigen or imrnunogen can be included.
  • an immune response to more than one antigen can be induced by immunization.
  • antigens or immunogens include, but are not limited to, tumor antigens of other tumor antigens from FGF-5-expressing cancers which may increase or provoke CD4 + T-cell (helper T-cell) responses supportive of an FGF-5 immune response.
  • additional amino acid sequences can be of varying length, such as at least about 5 amino acids, at least abut 10 amino acids, at least about 25 amino acids, at least about 50 amino acids, at least about 100 amino acids, or no more than about 500 amino acids, such as no more than about 250 amino acids, no more than about 100 a ino acids, no more than about 75 amino acids, no more than about 50 amino acids, no more than about 25 amino acids, no more than about 15 amino acids, or no more than about 10 amino acids.
  • the epitopes in the composition can be identical or different, and together they may provide equivalent or greater biological activity than the parent epitopes(s).
  • multiple epitopic peptides can be combined in a "cocktail" to provide enhanced immunogenicity, and peptides can be combined with peptides having different MHC specificities.
  • Such compositions can be used to effectively broaden the immunological coverage provided by therapeutic, immune stimulatory composition or diagnostic methods and compositions.
  • epitopic peptides are linked with or without a spacer molecule to form polymers (multimers), or can be formulated in a composition without linkage, as an admixture. Where the same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, heteropolymers with repeating units are provided.
  • Linkages for homo- or hetero-polymers or for coupling to carriers and adjuvants can be provided in a variety of ways, such as through covalent linkages between epitopic peptides or noncovalent linkages capable of forming intermolecular and intrastructural bonds.
  • the spacer can include relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions and may have linear or branched side chains.
  • Particular examples of spacers include on or more alanine or glycine residues, or other nonpolar amino acids or neutral polar amino acids.
  • Spacers can be either homo- or hetero-oligomers and can include one or two residues, more typically three to six residues.
  • Spacers can be attached to epitopic peptides at the C-terrninus, N-terminus or a side chain of one or more of the amino acids.
  • crosslinking agents that can be used to interconnect a plurality of epitopes include crosslinking agents having an aldehyde (such as glutaraldehyde), carboxyl, amine, amido, imido or azidophenyl functional group.
  • aldehyde such as glutaraldehyde
  • carboxyl such as glutaraldehyde
  • amine amido
  • imido or azidophenyl functional group such as butyraldehyde is used as a crosslinking agent, a divalent imido ester or a carbodiimide.
  • cysteine residues can be added at the amino- and carboxy- ter ini to permit formation of bonds between peptides via controlled oxidation of the cysteine residues.
  • Heterobifunctional agents which generate a disulfide link at one functional group end and a peptide link at the other, including N-succidimidyl-3-(2- pyridyldithio) proprionate (SPDP) can also be employed.
  • SPDP N-succidimidyl-3-(2- pyridyldithio) proprionate
  • SPDP N-succidimidyl-3-(2- pyridyldithio) proprionate
  • a variety of such disulfide/amide forming agents are known (For example, Immun. Rev. 62:185, 1982).
  • Other bifunctional coupling agents form a thioether rather than a disulfide linkage.
  • thioether forrning agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid, 2-iodoacetic acid, and 4-(N- maleimido-methyl) cyclohexane-1-carboxylic acid.
  • the carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4- sulfonic acid, sodium salt.
  • One coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
  • the linkage does not substantially interfere with the immunogenicity of the linked epitopic peptides.
  • a particular example of a fusion protein which includes one or more FGF-5 HLA-A2 or -A3 epitopic peptide sequences, can be used to present the epitopic peptides to a subject.
  • a recombinant HBV surface antigen protein is prepared in which the HBenv amino acid sequence is altered so as to more effectively present epitopes of peptide regions described herein to stimulate an immune response.
  • a polypeptide may incorporate several epitopes. Coding sequences for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185, 1981).
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein as disclosed herein and known in the art.
  • the disclosed epitopes such as an FGF-5 HLA-A2 eptiope, for example SEQ ID NO: 7, is obtained from natural sources.
  • an FGF-5 protein is subjected to selective proteolysis, for example by splitting the protein with chemical reagents or enzymes.
  • the disclosed epitopes such as an FGF-5 HLA-A3 eptiope, for example SEQ ID NO: 1-6, 9 or 17, are relatively short, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic peptide synthesizers are commercially available and can be used in accordance with known protocols. Chemical synthesis of peptides is described in: S. B. H.
  • recombinant DNA technology can be employed wherein a nucleotide sequence which encodes one or more epitopic peptides is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression, as disclosed herein.
  • the length of the amino acid sequence produced can depend on the method of producing the sequence. If the sequence is made by assembling amino acids by chemical means, the sequence ideally does not exceed, for example, about 50, about 40, or about 30 amino acids. If the synthetic peptide is made by translating a nucleic acid, the peptide can be any length, including, for example, about 100 amino acids or more. However, the peptide can also be shorter, for example, no more than 50, no more than 40, no more than 20, no more than 10, no more than 9, or no more than 8 amino acids.
  • FGF-5 HLA-A2 or HLA- A3 peptides (and nucleic acids encoding such epitopes), such as SEQ ID NOS: 1-7, 9 or 17, and variants, fragments, fusions, and mixtures thereof, can be tested for their potential as an immunogenic molecule(s) or compositions with binding assays, in vitro cell culture techniques and in small mammal models.
  • Proliferative assays can be used to measure the ability of the disclosed FGF-5 peptides to stimulate a T-cell response (PCT publication WO 02/22860).
  • T-cells (2 x 10 4 ) or irradiated peripheral blood mononuclear cells (5 x 10 4 ) are seeded, in duplicate, into wells with or without about 200 ⁇ g/ml peptide (Hemmer, et al, 1998, J. Pept. Res. 52:338-45). Proliferation is measured by 3 H-thymidine incorporation (Hemmer et al, 1991, J. Exp. Med. 185:1651- 9).
  • a therapeutic composition that includes an FGF-5 HLA-A2 or HLA- A3 peptide is one that can increase a T-cell response (proliferation) by at least about 10%, for example at least about 20%, or even about 50%, as compared to an amount of proliferation in the absence of the HLA-A2 or HLA- A3 peptide.
  • FGF-5 HLA-A2 and HLA-A3 peptides can also be tested in a cytotoxic T lymphocyte (CTL) assay (see, for example, Sette et al, 1994. J. Immunol. 153:5586- 92, and PCT publication WO 01/55177). Briefly, the spleen of peptide immunized transgenic mice are collected aseptically 10 days after immunization and placed in 5 ml of cell medium (RMPI 1640, penicillin +streptomycin, 2% Hepes buffer, 10% Fetal calf serum) on ice.
  • CTL cytotoxic T lymphocyte
  • splenocytes are cultured for 6 days in the presence of LPS blasts coated with 100 ⁇ g/ml of the peptide (stimulator cells) and then assayed for peptide- specific CTL activity by using EL4-A2 and EL4 cell lines in the presence or absence of the query peptides.
  • a therapeutic composition that includes an FGF-5 HLA-A2 or HLA- A3 peptide is one that can increase peptide-specific CTL activity by at least about 10%, for example at least about 20%, or even about 50%, as compared to an amount of CTL activity in the absence of the HLA-A2 or HLA- A3 peptide.
  • the disclosed peptides can also be used as diagnostic reagents.
  • an FGF-5 HLA-A2 or HLA-A3 epitopic peptide, or combinations thereof can be used to determine the susceptibility of a particular individual to a treatment regimen that employs the peptide or related peptides.
  • diagnostic reagents epitopic peptides can be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual.
  • EXAMPLE 1 Identification of an HLA-A3-Restricted Epitope This example describes methods used to identify the HLA- A3 -restricted epitope recognized by Clone 2. Truncated fragments of the FGF-5 gene (nucleic acid cDNA sequence, Genbank Accession No: M37825 (SEQ ID NO: 15); amino acid sequence, Genbank Accession No: AAB06463 (SEQ ID NO: 16)) were cloned into the expression vector pMEl 8S and tested by transfection into HLA-A3 expressing COS7 and recognition by Clone 2 assessed as follows.
  • Clone 2 CTL was assessed by culturing COS cells transfected with the FGF-5 sequences shown in FIG. 1 (150 ng plasmid was used to transfect 5 x 10 4 COS-A3 cells/well using Lipofectamine) in 96-well plates. After an overnight culture, 2 x 10 4 cells/well of Clone 2 CTL were added. After incubating for 20-24 hours, supernatants were harvested and IFN- ⁇ concentration was analyzed by ELISA (Endogen, Woburn, MA), where IFN- ⁇ concentration is considered proportional to CTL activation.
  • ELISA Endogen, Woburn, MA
  • ELISA plates (96 well flat bottom, Costar, NY) were coated with anti-human IFN- ⁇ monoclonal antibody (2G1, ENDOGEN, MA) at 1 ⁇ g/ml, 100 ⁇ l/well overnight. After washing, plates were blocked with PBS-5% FBS (fetal bovine serum) for one hour (200 ⁇ l/well), the samples added (50 ⁇ l/well) and incubated for 90 minutes. Subsequently, the plates were washed and biotin-labeled anti-human IFN- ⁇ monoclonal antibody (B 133.5, ENDOGEN, MA) was added at 0.5 ⁇ g/ml, 50 ⁇ l/well, and incubated for one hour.
  • 2G1, ENDOGEN, MA anti-human IFN- ⁇ monoclonal antibody
  • the plates were subsequently washed and 2000-times diluted HRP-streptavidin conjugate (Zymed Laboratories, CA) was added and incubated for 30 minutes. Plates were washed and DAKO TMB One-Step Substrate System (DAKO Corporation, CA) was added (100 ⁇ l/well). The coloration reaction was stopped by adding 0.18 M sulfuric acid (100 ⁇ l/well) and the optical density at 450nm wave-length was measured. Recombinant human IFN- ⁇ (Endogen, MA) was used as a standard.
  • the shortest stimulatory fragment recognized (G8) encoded 60 amino acids (SEQ ID NO: 15), indicating that the A3 epitope resided between amino acids 161 and 220 of FGF-5 (Genbank Accession No: AAB06463). Further truncation of 36 nucleotides from the 5' end or 24 bases from 3 1 end of this minimal coding sequence obviated recognition by Clone 2.
  • HLA Class I binding peptides are typically 8-10 a ino acids in length. Therefore, stepwise internal deletions were made within the mini-gene encoding the 60 amino acid fragment. Internal deletions of 30 nucleotides at a time demonstrated that removal of FGF-5 amino acids 181-190 (but not 191-200) resulted in a sequence that was recognized when transfected as a gene into an HLA- A3 target or when pulsed as a synthetic peptide on HLA-A3 EBV-B cells. Transfection and reactivity were assessed as described above.
  • Pulsing of synthetic peptide involved co-incubation of EBV-B cells or dendritic cells (HLA-A3 + as well as HLA- A3 " controls) with high levels of various synthetic peptides from FGF-5 (at 1 nM-10 uM) in medium for 30-180 minutes. These cells were then either assayed or washed and assayed by the addition of Clone 2 T-cells for 24 hours and assay of supernatant IFN- ⁇ by ELISA. Stepwise internal deletions were made, starting with a recognized mini-gene to identify residues important for epitope recognition. As shown in FIG.
  • plasmid G41 encoding FGF-5 amino acids 172-176 and 199-220, was the smallest construct that was recognized as strongly as full-length FGF-5. However, some of the other deletion mutants retained immunogenic activity, just not a strongly as full-length FGF-5.
  • mini-genes with single alanine substitutions at each of the 27 codons within G41 were made to demonstrate the contribution of each amino acid to recognition.
  • Point mutations were introduced by Quick Change Site-Directed Mutagenesis kit from Stratagene following the manufacture's protocol. As shown in FIG. 3, important residues lay at the two termini of the peptide at positions 172 (N 172 ), 174 (Y 174 ), 176 (S 176 ), and 217-220 (P 217 , R 218 , F 219 , and K 220 ) (with potential lesser contributions from amino acids 200 and 214).
  • Peptide pulsing was done in RPMI medium 1640 (RPMI) and the co-culture was done in RPMI with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Peptides were synthesized either on the Pioneer Peptide Synthesizer (PE Biosystem) or on the AMS 222 multiple peptide synthesizer (Gilson) using standard F-moc chemistry. The molecular weights of peptides were verified by mass spectrometry (Bio- Synthesis Inc.). As shown in FIG.
  • This example describes the methods used to demonstrate the mechanism that generates the 9-mer fusion peptide (SEQ LO NO: 1) from the FGF-5 gene.
  • RNA splicing was eliminated as an explanation because numerous internal truncation mutants recognized by C2 (FIG. 2, construct G26-29 and G34-41) possess different sequences flanking potential splice sites, and because neither the original FGF-5 sequence nor any of the truncation mutants share splicing motifs.
  • FIG. 6 To eliminate the possibility of ribosome skipping, three plasmids with termination codons inserted between the two determinant-encoding fragments (FIG. 6) were generated using standard molecular biology methods, and the ability of these sequences to activate Clone 2 determined as described above. As shown in FIG. 7, no plasmids sensitized cells for Clone 2 recognition following transfection into COS-A3 APCs. This result also refutes epitope generation by aberrant RNA splicing because it appears that the intervening sequence between the two determinant-encoding fragments is transcribed and translated, indicating a post-translational mechanism.
  • HLA- A3 epitope SEQ ID NO: 1
  • EBV transformed B cells EBV transformed B cells
  • FIGS. 8B and 8C are not the result of contamination of the 49-mer peptide (SEQ ID NO: 13) with 9-mer fusion peptide (SEQ ID NO: 1).
  • Peptide solutions (from ⁇ 4xl 0 9 cells, ⁇ 1 ,200 ml each) were prepared and were concentrated by Sep-pak Plus C 18 column (Waters). After lyophilization and resolubilization into 200 ⁇ l of 5% acetonitrile 0.05% (v/v) TFA, peptides were fractionated using a C 18 HPLC column (218TP54, Grace Vydak) between 5% acetonitrile with 0.05% (v/v) TFA and 40% acetonitrile with 0.05% (v/v) TFA, linear gradient (1 % and 1 ml/min) .
  • HPLC also demonstrated that the 9-mer (SEQ LD NO: 1) represents the naturally processed peptide from FGF-5.
  • SEQ LD NO: 1 represents the naturally processed peptide from FGF-5.
  • FIG. 10D antigenic activity of acid- stripped peptides from COS-A3 cells expressing FGF-5 was exclusively recovered from fraction 11.
  • none of the fractions from non-FGF-5 expressing cells demonstrated significant antigenic activity (FIC. IOC).
  • FGF-5 determinant is the covalently-linked 9-mer (SEQ ID NO: 1) and not the 49-mer (SEQ ID NO: 13) nor the non-covalently bonded peptides NTYAS (amino acids 1-5 SEQ ID NO: 1) and PRFK (amino acids 6-9 SEQ ID NO: 1), which should elute in fractions 28 and 5 respectively.
  • the result is to create a tumor target capable of presenting not only its autologous antigens to Clone 2, but also class I and Il-restricted determinants from gplOO to control T-cell clones which recognize this melanoma antigen (class II gplOO recognition is restricted by DR ⁇ 1 *0401 , naturally present on the RCC).
  • Transduced RCC cells were treated with the citric acid buffer (pH 3.1) for 90 seconds, washed, then cultured in 10 ⁇ M clasto-Lactacystin ⁇ -Lactone (Calbiochem) for 3 hours. After washing, T-cells were added and IFN- ⁇ was measured at 20 hours using the methods described in Example 1.
  • a CD8 + T cell clone that recognizes the gpl00 20 -217 determinant was used as a positive control for inhibitor effectiveness and a CD4 + T cell clone that recognizes gpl00 44-59 determinant was used as a control to eliminate non-specific effects of inhibitors on general antigen presentation capacity.
  • MHC class I-restricted peptides As shown in FIGS. 11 A and 1 IB, presentation of both MHC class I-restricted peptides was blocked by the proteasome inhibitor clasto-Lactacystin ⁇ -lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport. MHC class Il-restricted gplOO recognition was unaffected.
  • NTYASPRFK FGF-5 HLA-A3 peptide (SEQ ID NO: 1) is generated by protein splicing from longer biosynthetic or synthetic precursors.
  • the splicing observed may likely occur via reverse proteolysis, as described for concanavalin A (Carrington et al. 1985. Nature 313: 64-7; Min and Jones. 1994. Nat. Struct. Biol. 1:502-4).
  • the proteasome and TAP-dependence of antigen presentation and the successful splicing of plasmid encoded trunctated FGF-5 based-polypeptides lacking leader sequences indicate that splicing occurs in cytosol. In the presence of the leader sequence on FGF-5, delivery of FGF-5 to the cytosol for splicing may require the endoplasmic reticulum associated degradation pathway.
  • an HLA-A2-restricted epitope of FGF-5 was identified using the following methods.
  • Peripheral blood lymphocytes (PBL) from HLA-A2 subjects were stimulated with FGF-5 peptides. If peptide reactivity was generated in vitro, these peptide-reactive T-cell cultures were tested for recognition of FGF-5 expressing, HLA-A2 positive tumors. Synthetic peptide preparations were used for approximately a dozen candidate peptides. Using one preparation, 5194 (synthesized by Macromolecular Resources, Ft.
  • a candidate A2-binding 10-mer peptide was present within this region (encoded by nucleotides 511-540 of Genbank Accession No: M37825, corresponding to amino acids 117-126 of Genbank Accession No: AAB06463) and a purified synthetic preparation of this peptide, MLSVLEIFAV (SEQ ID NO: 7), was well recognized by tumor-reactive CTL clones when pulsed onto T2 cells in nanomolar amounts (FIG. 13).
  • MLSVLEIFAV (SEQ ID NO: 7) is an FGF-5 HLA- A2 epitope
  • site-directed mutagenesis was used to convert A to C at nucleotide 516 (of Genbank Accession No: M37825; SEQ ID NO: 15) which resulted in a substitution of phenylalanine for the native leucine at the HLA-binding anchor residue (LI 18F) at position two of SEQ LD NO: 7.
  • This modified gene product (MFSVLEIFAV; SEQ ID NO: 8) was no longer recognized by the CTL clones when transfected into HLA-A2 + target cells, demonstrating that Leu 118 is important for recognition by tumor-reactive T-cell clones.
  • HLA-A2-binding substitutions can be made at possible anchor residues, such as amino acid Leu 118, and amino acid Val 126, to improve immunogenicity of this epitope.
  • NTYASPRFK SEQ ID NO: 1
  • peptides or variants or fusions thereof can be used as an immunotherapy for subjects with an FGF-5 expressing or overexpressing tumor, such as an adenocarcinoma.
  • the present disclosure provides methods for immune stimulation (for example vaccination) using these peptides to enhance the number of FGF-5-reactive CTL precursors in subjects with an FGF-5 expressing or overexpressing tumor, such as RCC, or affect the anticipated response rate from high-dose IL-2.
  • Subjects with advanced clear cell renal cancer expressing FGF-5 are administered an HLA-appropriate peptide vaccination (SEQ ID NO: 7 for HLA-A2 + subjects or SEQ ID NO: 1 for HLA-A3 + subjects) emulsified in incomplete Freund's Incomplete Adjuvant (IF A) in three cohorts.
  • Cohort A includes subjects with measurable metastatic disease who do not have an indication for immediate IL-2 therapy. They receive vaccination every three weeks with clinical response and immunological response to vaccination as primary endpoints. If tumor progression is documented, subjects eligible to receive high-dose IL-2 receive it along with continued peptide vaccine.
  • Cohort B is designed to include subjects with measurable metastatic disease who have indications for immediate IL-2 therapy.
  • Cohort C includes subjects who have had high-risk primary tumors removed. They receive the same HLA-appropriate peptide vaccination described above for up to one year or until tumor relapse is documented. Immunological response is the primary endpoint for this cohort.
  • a variety of methods are used to administer the disclosed immunogenic peptides.
  • the methods of administration can be tailored, for example to the condition of the subject.
  • subjects are divided into cohorts with measurable metastatic disease (Cohorts A and B) or high-risk loco-regional disease (Cohort C).
  • Subjects with measurable metastatic disease are separated into those that receive immediate IL-2 therapy (Cohort B) or those who do not (cohort A) as determined by tumor burden, tempo of disease or other co-morbidities.
  • Subjects in Cohort B who receive immediate IL-2 therapy begin with the same standard high-dose bolus IL-2 therapy in two cycles within every two month period, with each cycle preceded by a peptide + IFA vaccine the day prior to starting each IL-2 cycle. Because subjects with metastatic RCC are likely to progress within a few months (unless vaccination alone has an early and dramatic effect) and because starting vaccination and peptide simultaneously may blunt the ability to measure immune response in the blood, it is desirable to have a subject population who can safely undergo a more prolonged period of vaccination without receiving IL-2. However, methods of administration also include concurrent administration of IL-2.
  • Cohort C includes subjects who have undergone resection of either T3/T4 or N1/N2 primary tumors (i.e. Stage III disease) within six months of beginning therapy. These subjects undergo the same HLA- appropriate vaccination with peptide and IFA every three weeks and continue for up to one year or until disease relapse is documented. At the time of relapse, eligible subjects in Cohort C receive standard treatment with high-dose bolus IL-2 and continuing peptide vaccination.
  • HLA-A2 or HLA- A3 there are subgroups of subjects who are HLA-A2 or HLA- A3 (subjects who have both alleles are treated with an HLA- A3 peptide, SEQ ED NO: 1, or can be administered both peptides as described in Example 10).
  • HLA-A2 subjects are vaccinated subcutaneously with 1 mg of MLSVLEIFAV peptide (SEQ ID NO: 7) emulsified in IFA and HLA-A3 subjects receive 1 mg of the NTYASPRFK peptide
  • Table 3 Examples of Treatment Methods (Each cohort has subgroups for HLA- A2 and HLA-A3)
  • HLA-A2-restricted FGF-5 peptide FGF-5: 117-126 (SEQ ID NO: 7) and HLA- A3-restricted FGF-5 peptide: FGF-5: 172-176 + 217-220 (SEQ ID NO: 1) were produced to GMP grade by solid phase synthesis techniques by Multiple Peptide
  • the finished injectable dosage form is supplied as a 5 ml clear molded glass, siliconized vial containing 1.5 ml of a clear sterile solution. Each mL contains 1 mg of each peptide; 0. IN HC1 has been added to adjust pH. The vials are stored at -10 to -20°C until use. The peptides are used within 3 hours after thawing. The desired peptide is reconstituted and injected as an emulsion with IFA (NSC
  • Incomplete Freund's Adjuvant (Seppic, Inc.) is used as the Freund's Incomplete Adjuvant (IFA) (Fairfield, NJ). It is provided as an amber glass ampule containing 3 ml of a mineral oil solution based on mannide oleate (Montanide ISA-51). At the time of injection, peptide is mixed with the Montanide ISA.51 as described above.
  • adjuvants can be used, for example, Freund's complete adjuvant, B30- MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole ly pet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
  • Interleukin-2 Intravenous Administration
  • IL-2 (Chiron Corp., Emeryville, CA) is administered at a dose of 720,000 IU/kg as an intravenous bolus over a 15 minute period every eight hours beginning on the day after immunization and continuing for up to 5 days. Doses can be skipped depending on subject tolerance. Doses are skipped if subjects reach Grade III or IV toxicity due to IL-2 except for the reversible Grade III toxicities common to IL-2 such as diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and laboratory changes as detailed in Table 3. If this toxicity is easily reversed by supportive measures then additional doses can be given.
  • IL-2 is provided as a lyophilized powder.
  • the vial is reconstituted with 1.2 ml of sterile water for injection, USP, and the resultant concentration is 18 million IU/ml. Since vials contain no preservative, reconstituted solution is used with 8 hours.
  • Reconstituted IL-2 is further diluted with 50 ml of 5% human serum albumin (HSA).
  • HSA human serum albumin
  • the HSA is added to the diluent prior to the addition of RIL-2. Dilutions of the reconstituted solution over a 1000-fold range (that is, 1 mg/ml to 1 mcg/ml) are acceptable in either glass bottles or polyvinyl chloride bags. Ideally, IL-2 is not mixed with saline-containing solutions.
  • a subject's response to treatment is evaluated by a complete blood count, acute care, hepatic and mineral panels every three weeks.
  • Subjects receiving IL-2 in the hospital obtain a CBC, acute care, hepatic and mineral panel evaluated every 1 to 2 days of treatment.
  • Peptide immunizations can be aciministered as an outpatient, unless inpatient admission is indicated for treatment of vaccine-related side-effects or underlying disease management.
  • Biopsies of tumor tissue or lymph node can be performed, but are not required during the course of therapy. Apheresis is performed prior to the first and third immunizations and three weeks after the last immunization. If not possible due to scheduling or vascular access issues, collection of 50 ml of peripheral blood can be substituted.
  • PBL peripheral blood lymphocytes
  • FGF-5-specific CTL can then be tested by cytokine release assay, or ELISPOT assay using tumor, FGF-5- transfected or peptide-loaded target cells and compared to pre-treatment PBMC to determine immune response to vaccination, as previously described (Id.). In general, differences of 2 to 3 fold in these assays are indicative of true biologic differences.
  • subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of peptide that they previously received for up to a year of total therapy.
  • a mixed response is the shrinkage of some lesions but an increase in others. Subjects with mixed responses may only receive treatment for an additional 2-3 months without showing true disease stability or a bona fide minor or major response (i.e. no further progression). A maximum of two re-treatment cycles can be given following a complete response.
  • measurable lesions up to a maximum of 10 lesions, representative of all involved organs are identified as target lesions and recorded and measured at baseline.
  • Target lesions are selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repetitive measurements (either by imaging techniques or clinically).
  • a sum of the longest diameter (LD) for all target lesions is calculated and reported as the baseline sum LD.
  • the baseline sum LD is used as reference to further characterize the objective tumor response of the measurable dimension of the disease as outlined in Table 4.
  • the best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started).
  • the subject's best response assignment can depend on the achievement of both measurement and confirmation criteria as shown in Table 6.
  • the duration of overall response is measured from the time measurement criteria are met for CR/PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started).
  • the duration of overall complete response is measured from the time measurement criteria are first met for CR until the first date that recurrent disease is objectively documented.
  • Examples 4-9 describe methods that can be used to administer SEQ ID NOS: 1 or 7 (FGF-5 HLA- A3 or -A2 epitope, respectively) to subjects having an FGF-5 expressing or over-expressing tumor, and who are HLA-A2 + or HLA-A3 + .
  • FGF-5 HLA-A3 or -A2 epitopes can be administered, such as variants, fragments, or fusions of SEQ ID NO: 1 or 7, for example SEQ ID NOS: 3-6, 9, and 17.
  • the present disclosure includes administration of a combination of an FGF-5 HLA- A3 epitope and an FGF-5 HLA-A2 eptiope to a subject, such as a subject who is both HLA-A2 + and HLA-A3 + .
  • a combination of FGF- 5 HLA- A3 and -A2 epitopes are administered to a subject at a therapeutically effective dose.
  • subjects having both an HLA-A2 + and HLA-A3 + allele are vaccinated subcutaneously with 1 mg of an FGF-5 HLA-A2 peptide (such as SEQ ID NO: 7 or a variant, fragment, or fusion thereof) and 1 mg of an FGF-5 HLA- A3 peptide (such as SEQ ID NO: 1, 9, 17, or a variant, fragment, or fusion thereof) in IFA.
  • an FGF-5 HLA-A2 peptide such as SEQ ID NO: 7 or a variant, fragment, or fusion thereof
  • an FGF-5 HLA- A3 peptide such as SEQ ID NO: 1, 9, 17, or a variant, fragment, or fusion thereof
  • FGF-5 HLA-A3 and-A2 peptides In addition to the FGF-5 HLA-A3 and-A2 peptides, other agents can be administered to the subject, such as IL-2. Following administration of the FGF-5 HLA-A2 and -A3 peptides, the FGF-5 expressing or overexpressing tumor can be monitored for regression.
  • a ⁇ missering the FGF-5 HLA-A2 and HLA- A3 epitopes of the present disclosure can be accomplished by any means known to the skilled artisan.
  • a pharmaceutically acceptable carrier can be provided for the FGF-5 HLA-A2 and HLA-A3 epitopes disclosed herein.
  • a pharmaceutically acceptable carrier may not be required to induce an immune response to the FGF-5 HLA-A2 or HLA- A3 epitope.
  • pharmaceutically acceptable carriers include, but are not limited to, substances that are animal, vegetable, or mineral in origin, that are physiologically acceptable and function to present the FGF-5 HLA-A2 or HLA-A3 epitope to the immune system.
  • a wide variety of pharmaceutically acceptable carriers are acceptable, and include materials which are inert, those having biological activity, or those that promote an immune response.
  • a particular example of a pharmaceutically acceptable carrier is an agent that aids in stimulation of the immune response, such as an adjuvant.
  • Adjuvants are nonspecific immune stimulators that increase the immune readiness and aid in stimulating a higher level (titer) of serum antibodies that recognize the epitopic peptide sequences.
  • Adjuvants include, for example, Freund's complete adjuvant, Freund's incomplete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
  • the adjuvant is a lipid it may be linked to the epitopic peptide(s).
  • a high titer of antibodies serves to protect a subject from the tumor to which the antibodies are directed.
  • Other examples of pharmaceutically acceptable carriers include physiologically acceptable masses to which the eptiope it attached, and in some examples, enhances the immune response.
  • a mass is one or more amino acids or other moieties, such as a dimer, oligomer, or higher molecular weight polymer of a sequence of amino acids of an FGF-5 HLA-A2 and HLA- A3 epitope.
  • an FGF-5 HLA-A2 or HLA-A3 epitope can be formed from naturally available materials or synthetically produced and can then be polymerized to build a chain of two or more repeating units so that the repeating sequences form both the carrier and the immunogenic polypeptide.
  • additional amino acids can be added to one or both ends of an FGF-5 HLA-A2 and HLA- A3 epitope.
  • Polysaccharides can also attached to the disclosed epitopes, and include those of molecular weight 10,000 to 1,000,000, such as starches, dextran, agarose, ficoll, or its carboxyl methyl derivative and carboxy methyl cellulose.
  • Polyamino acids can also attached to the disclosed epitopes, and include, polylysine, polyalanyl polylysine, polyglutamic acid, polyaspartic acid and poly (C 2 -C 10 ) amino acids.
  • Organic polymers can also attached to the disclosed epitopes, and these polymers include, for example, polymers and copolymers of amines, amides, olefins, vinyls, esters, acetals, polyamides, carbonates and ethers and the like. Generally speaking, the molecular weight of these polymers will vary dramatically. The polymers can have from two repeating units up to several thousand, such as two thousand repeating units.
  • the number of repeating units will be consistent with the use of the immunizing composition in a host animal.
  • such polymers have a lower molecular weight, for example, between 10,000 and 100,000 kD (the molecular weight being determined by ultracentrifugation).
  • Inorganic polymers can also be employed. These inorganic polymers can be inorganic polymers containing organic moieties. In particular, silicates and aluminum hydroxide can be used as carriers. Ideally, the carrier is one which is an immunological adjuvant. In such cases, it is contemplated that the adjuvant be muramyl dipeptide or its analogs.
  • FGF-5 HLA-A2 or HLA- A3 eptitopic peptides are administered to a subject in a colloidal dispersion system.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. Large uni-lamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al, 1981, Trends Biochem. Sci. 6:77, 1981).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamme, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs that contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system can be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • a liposome includes the desired peptide and is directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic epitope.
  • Particular lipid residues such as palmitic acid or other uncharged fatty acid residues of different chain lengths and degrees of unsaturation, ranging from acetic to stearic acid as well as to negatively charged succinyl residues may be attached to the epitopic via the appropriate carboxylic acid anhydrides.
  • the lipids can be directly attached to the epitopic peptide or indirectly through a linkage as described above.
  • a lipid can be attached directly to the amino terminus of the peptide or via a linkage such as Ser-Ser, Gly, Gly-Gly, or Ser.
  • various viral vectors can be utilized. These vectors include, but are not limited to, adenovirus, herpes virus, vaccinia, or an RNA virus such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • a vector such as the gibbon ape leukemia virus (GaLV) can be utilized.
  • GaLV gibbon ape leukemia virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • a nucleic acid sequence encoding an FGF-5 HLA-A2 or HLA- A3 peptide into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific.
  • Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein.
  • Prefened targeting is accomplished by using an antibody to target the retroviral vector.
  • an antibody to target the retroviral vector.
  • Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the polynucleotide encoding an FGF-5 HLA-A2 or HLA- A3 epitope.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317, and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • compositions disclosed herein can be prepared and administered in dose units.
  • Solid dose units include tablets, capsules, transdermal delivery systems, and suppositories.
  • For treatment of a subject depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of a therapeutic amount can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • the FGF-5 epitopes and pharmaceutical compositions disclosed herein can be administered by any method used in the art, for example locally or systemically, such as topically, intravenously, orally, parenterally or as implants.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • compositions are suitable for use in a variety of drug delivery systems.
  • Amounts effective for therapeutic use can depend on the severity of the disease and the age, weight, general state of the patient, and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models maybe used to determine effective dosages for treatment of particular disorders.
  • the dose range for an FGF-5 HLA-A2 or HLA- A3 epitope protein is from about 0.1 ⁇ g/kg body weight to about 100 mg/kg body weight.
  • Other suitable ranges include doses of from about 1 ⁇ g/kg to 10 mg/kg body weight.
  • the dose is about 1.0 ⁇ g to about 50 mg, for example, 1 ⁇ g to 1 mg, such as 1 mg peptide per subject.
  • the dosing schedule can vary from daily to as seldom as once a year, depending on clinical factors, such as the subject's sensitivity to the peptide and tempo of their disease. Therefore, a subject can receive a first dose of immunogenic FGF-5 HLA-A2 or HLA-3 epitope, and then receive a second dose (or even more doses) at some later time(s), such as at least one day later, such as at least one week later.
  • initial immunization can be followed by boosting dosages of from about 1 ⁇ g to 50 mg, for example, 1 ⁇ g to 500 ⁇ g, such as 1 ⁇ g to about 250 ⁇ g of peptide.
  • a boosting regimen can be followed over weeks to months, depending upon the patient's response and condition by measuring specific immune activity in the patient's blood.
  • it can be preferable to administer doses such as those described above by alternate routes including intravenously or intrathecally. Continuous infusion may also be appropriate.
  • purified therapeutically active molecules are combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the peptides and nucleic acids herein disclosed.
  • the nature of the pharmaceutically acceptable carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • compositions can contain only one type of therapeutic molecule, or can include a combination of several types of therapeutic molecules, such as other anti-neoplasic agents, such as IL-2.
  • protein-based pharmaceuticals may be only inefficiently delivered through ingestion.
  • pill-based forms of pharmaceutical proteins can be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow-release formulations may be produced by combining the target protein with a biocompatible matrix, such as cholesterol.
  • Another possible method of aclministering protein pharmaceuticals is through the use of mini osmotic pumps. As stated above a biocompatible carrier would also be used in conjunction with this method of delivery.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally about 10-95% of active ingredient, that is, one or more FGF-5 epitopes disclosed herein, for example, at a concentration of about 25%- 75%.
  • the disclosed epitopic peptide compositions can be • supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01% - about 20% by weight, for example, about 1% - about 10%.
  • the surfactant ideally is nontoxic and soluble in the propellant.
  • Representative surfactants include 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.
  • the surfactant can constitute about 0.1% - about 20% by weight of the composition, for example, about 0.25 - about 5% by weight of the composition.
  • the balance of the composition is typically propellant.
  • a carrier can also be included, as desired. For example, lecithin may be used for intranasal delivery.
  • the disclosed FGF-5 antigenic epitope sequences can be modified, while retaining an ability to generate an immune response, such as in a subject in whom the peptide is administered.
  • exemplary modifications include, but are not limited to, FGF- 5 antigenic epitope analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) that generate an immune response.
  • the disclosed peptides include a sequence of amino acids, which can be either L- and/or D- amino acids, naturally occurring and otherwise.
  • FGF-5 antigenic epitope peptide sequences can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a - ⁇ ester, or converted to an amide of formula NRiR 2 wherein R and R 2 are each independently H or Ci-Ci ⁇ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to -Ci ⁇ alkyl or dialkyl amino or further converted to an amide.
  • Hydroxyl groups of the peptide side chains maybe converted to d-C 16 alkoxy or to a Ci-Ci ⁇ ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with -C ⁇ alkyl, CrC 16 alkoxy, carboxylic acids and. esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C 2 -C 4 alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • Peptidomimetic and organomimetic embodiments are also within the scope of the present disclosure, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins disclosed herein having measurable or enhanced ability to bind an antibody.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD).
  • a desired response is stimulation of a CTL response to an FGF-5 expressing or over-expressing tumor, such as RCC, resulting in halting or slowing the progression of, or inducing a regression of a pathological condition or which is capable of relieving signs or symptoms caused by the condition.
  • FGF-5 expressing or over-expressing tumor such as RCC
  • a therapeutic effect is regression of the tumor, lysis of the cells of the tumor, or both. Treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently.
  • the efficacy of IL-2 is increased by at least 10%, for example at least 20%, in the presence of another agent, as measured by a clinical response.
  • the therapeutically effective amount also includes a quantity of FGF-5 HLA- A3 or HLA-A2 epitope protein (such as SEQ ID NO: 1-7, 9 or 17, or variants or fragments thereof), autologous CTLs specific to FGF-5, or combinations thereof sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to improve signs or symptoms a disease such as cancer, for example by modulating, for example increasing a CTL response against a tumor expressing or overexpressing FGF-5.
  • CTLs specific to FGF-5 can be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the effective amount of can be dependent on the source applied (for example FGF-5 peptide isolated from a cellular extract versus a chemically synthesized and purified FGF-5 peptide, or a variant or fragment that may not retain full FGF-5 activity), the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • a therapeutically effective amount of FGF-5 HLA- A3 or HLA-A2 epitope protein can vary from about 0.01 mg/kg body weight to about 10 mg/kg body weight, such as about 1 mg per subject.
  • the general term "subject being treated” is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows) that require modulation of a CTL response against a tumor that is expressing or overexpressing FGF-5.

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Abstract

La présente invention se rapporte à des peptides que l'on utilise dans l'immunothérapie de tumeurs. Les peptides de cette invention sont dérivés de la séquence d'acides aminés d'un antigène associé au carcinome d'une cellule rénale, et du vecteur de croissance fibroplastique 5 (FGF-5). Selon une variante, le peptide est un épitope HLA-A3 (notamment NTYASPRFK). Selon une autre variante, le peptide est un épitope HLA-A2 (notamment MLSVLEIFAV). Les procédés de cette invention utilisent de tels peptides et leurs variants ou fusions afin de stimuler une réponse immunitaire chez un patient. Les peptides de cette invention peuvent être formulés sous forme de compositions pharmaceutiques en vue de leur administration chez un patient.
PCT/US2003/037065 1999-10-02 2003-11-19 Epitopes immunogenes pour facteur de croissance fibroplastique 5 (fgf-5) presente par hla-a3 et hla-a2 WO2004045555A2 (fr)

Priority Applications (4)

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CA002506684A CA2506684A1 (fr) 2002-11-19 2003-11-19 Epitopes immunogenes pour facteur de croissance fibroplastique 5 (fgf-5) presente par hla-a3 et hla-a2
AU2003298677A AU2003298677A1 (en) 2002-11-19 2003-11-19 Immunogenic epitopes for fibroblast growth factor 5 (fgf-5) presented by hla-a3 and hla-a2
EP03796431A EP1562622A2 (fr) 2002-11-19 2003-11-19 Epitopes immunogenes pour facteur de croissance fibroplastique 5 (fgf-5) presente par hla-a3 et hla-a2
US11/134,703 US20060009393A1 (en) 1999-10-02 2005-05-19 Immunogenic epitopes for fibroblast growth factors 5 (FGF-5)

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US42792002P 2002-11-19 2002-11-19
US60/427,920 2002-11-19

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025271A2 (fr) * 1999-10-02 2001-04-12 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Facteur de croissance des fibroblastes 5 (fgf-5) dans un antigene de leucocyte t associe aux tumeurs
US20020031805A1 (en) * 1999-12-29 2002-03-14 Conklin Darrell C. Novel FGF homolog zFGF10

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025271A2 (fr) * 1999-10-02 2001-04-12 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Facteur de croissance des fibroblastes 5 (fgf-5) dans un antigene de leucocyte t associe aux tumeurs
US20020031805A1 (en) * 1999-12-29 2002-03-14 Conklin Darrell C. Novel FGF homolog zFGF10

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