CA2506684A1 - Immunogenic epitopes for fibroblast growth factor 5 (fgf-5) presented by hla-a3 and hla-a2 - Google Patents
Immunogenic epitopes for fibroblast growth factor 5 (fgf-5) presented by hla-a3 and hla-a2 Download PDFInfo
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- CA2506684A1 CA2506684A1 CA002506684A CA2506684A CA2506684A1 CA 2506684 A1 CA2506684 A1 CA 2506684A1 CA 002506684 A CA002506684 A CA 002506684A CA 2506684 A CA2506684 A CA 2506684A CA 2506684 A1 CA2506684 A1 CA 2506684A1
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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]
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- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present disclosure relates to peptides for use in immunotherapy of tumor s. The peptides disclosed herein are derived from the amino acid sequence of a renal cell carcinoma-associated antigen, fibroblast growth factor-5 (FGF-5). In one example, the peptide is an HLA-A3 epitope (such as NTYASPRFK). In another example, the peptide is an HLA-A2 epitope (such as MLSVLEIFAV). Methods are provided for using such peptides, and variants or fusions thereo f, to stimulate an immune response in a subject. The peptides disclosed herein can be formulated into pharmaceutical composition for administration to a subject.
Description
IMMUNOGENIC EPITOPES FOR FIBROBLAST GROWTH FACTOR 5 (FGF-5) PRESENTED BY HLA-A3 AND HLA-A2 CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No.
60/427,920 filed November 19, 2002, herein incorporated by reference in its entirety.
FIELD
This application relates to human leukocyte antigen (HLA)-A3 and HLA-A2 epitopes of fibroblast growth factor 5 (FGF-5) and uses thereof.
BACKGROUND
Each year in the United States, approximately 30,000 patients are diagnosed with renal cell carcinoma (RCC) and approximately 12,000 patients die of this disease (Linehan et al., 2001. Cancers of the Genitourinary System. In Cancer:
Principles and Practice of Oncology. DeVita, Hellman, and Rosenberg, editors. Lippincott Williams and Wilkins, Philadelphia. 1343-149). Most patients initially present with either advanced local disease or metastatic disease. Metastatic disease has a dismal prognosis and even patients with advanced local disease are likely to develop metastases and die, despite successful nephrectomy.
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 ~0%
(Rosenberg et al., 199. Ann. Surg. 228:319; Fisher et al., 1997. Cancer J.
Sci. Amer.
3:570-S72). Therefore, new methods are needed to improve on IL-2 therapy and expand its curative potential to a greater percentage of patients with RCC.
This application claims the benefit of U.S. Provisional Application No.
60/427,920 filed November 19, 2002, herein incorporated by reference in its entirety.
FIELD
This application relates to human leukocyte antigen (HLA)-A3 and HLA-A2 epitopes of fibroblast growth factor 5 (FGF-5) and uses thereof.
BACKGROUND
Each year in the United States, approximately 30,000 patients are diagnosed with renal cell carcinoma (RCC) and approximately 12,000 patients die of this disease (Linehan et al., 2001. Cancers of the Genitourinary System. In Cancer:
Principles and Practice of Oncology. DeVita, Hellman, and Rosenberg, editors. Lippincott Williams and Wilkins, Philadelphia. 1343-149). Most patients initially present with either advanced local disease or metastatic disease. Metastatic disease has a dismal prognosis and even patients with advanced local disease are likely to develop metastases and die, despite successful nephrectomy.
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 ~0%
(Rosenberg et al., 199. Ann. Surg. 228:319; Fisher et al., 1997. Cancer J.
Sci. Amer.
3:570-S72). Therefore, new methods are needed to improve on IL-2 therapy and expand its curative potential to a greater percentage of patients with RCC.
Over the past ten years, rapid advances have been made in the understanding of the immune response to cancer in patients, largely through the study of melanoma.
Beginning with an initial description of an antigen on melanoma recognized by T-cells from a patient repetitively vaccinated with autologous tumor (van der Bruggen et al., 1991. Scieyace 254:1643-7), dozens of other melanoma-associated antigens have been identified which provoke T-cell responses (Boon et al., 1997. Immunol. Today 18:267-8; Robbins et al., 2000. Tumor Antigens Recognized by Cytotoxic Lymphocytes.
Ira Cytotoxic Cells: Basic Mechanisms and Medical Applications. Sitkovsky and Henkart, editors. J. B. Lippincott, Philadelphia. 363-383; Van den Eynde and B.P.van der. 1997.
Curr.Opin.Immunol.9:684-93).
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. Irnmuhol. 4:59-62). Such processed epitopes that bind to Class I MHC are typically 8-10 amino 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. 183:1131-40), protein production from anti-sense DNA strands (Van den Eynde et al., 1999. J. Exp. Med. 190:1793-1800), and post-translational modification of specific amino acids (Skipper et al., 1996. J.
Exp. Med.
183:527-34).
Augmented T-cell responses may improve IL-2 therapy. For example, 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 Ifast.
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).
Beginning with an initial description of an antigen on melanoma recognized by T-cells from a patient repetitively vaccinated with autologous tumor (van der Bruggen et al., 1991. Scieyace 254:1643-7), dozens of other melanoma-associated antigens have been identified which provoke T-cell responses (Boon et al., 1997. Immunol. Today 18:267-8; Robbins et al., 2000. Tumor Antigens Recognized by Cytotoxic Lymphocytes.
Ira Cytotoxic Cells: Basic Mechanisms and Medical Applications. Sitkovsky and Henkart, editors. J. B. Lippincott, Philadelphia. 363-383; Van den Eynde and B.P.van der. 1997.
Curr.Opin.Immunol.9:684-93).
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. Irnmuhol. 4:59-62). Such processed epitopes that bind to Class I MHC are typically 8-10 amino 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. 183:1131-40), protein production from anti-sense DNA strands (Van den Eynde et al., 1999. J. Exp. Med. 190:1793-1800), and post-translational modification of specific amino acids (Skipper et al., 1996. J.
Exp. Med.
183:527-34).
Augmented T-cell responses may improve IL-2 therapy. For example, 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 Ifast.
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).
These results have led to efforts to identify similar T-cells and tumor-associated antigens for IL-2 responsive tumors other than melanoma. However, this search has had limited success. A list of published tumor-associated antigens expressed by RCC is shown in Table 1. However, many of the RCC antigens shown in Table 1 are expressed in normal tissue, or are infrequently found when testing large numbers of individual renal cancers, making them less than ideal for tumor therapy. Other antigens appear to be poorly processed and presented by actual tumor cells (as opposed to target cells incubated with the [already processed] minimal peptide epitopes).
Table 1: Published RCC Antigens.
Antigen MHC Epitope ExpressionComments Reference Restriction on RCC
RAGE-1 B07 SPSSNRIRNT 2% Rarely on Gaugler RCC et al., 1996.
Immunogenetics 44:323-30 MN-CA 0201 HLSTAFARV 85% Difficult Vissers IX to detect et al., epitope 1999. Cancer presented on tumors* Res. 59:5554-9;
*Parkhurst, M
(personal communication) hTERT A0201 ILAKFLHWL 85% Some normalVonderheide et (teleomerase)03 KLFGVLRLK tissue expressional., 1999.
Imrnunity.
10:673-679 RU1 B51 VPYGSFKHV frequentUbiquitous Morel et on al., normal tissue2000. Immunity.
12:107-117 RU2 (AS)B07 LPRWPPPQL frequentAnti-sense Van den Eynde transcript et al., 1999.
Expressed JExp.Med.
in testis and normal 190:1793-800 kidney IntestinalB0702 SPRWWPTCL frequentAlt. ORF Ronsin epitope et al., carboxyl Expressed 1999. J
in esterase normal tissueInununol.
163:483-90 mut hsp70-20201 SLFEGIDIYT unique Tumor-specificGaudin et al., mutation 1999. J
Immurzol.
162:1730-8 M-CSF B3501 LPAWGLSPGEQEY60% Alt. ORF Probst-Kepper epitope et Expressed al., 2001.
in normal kidneyJExp.Med.
and liver 193:1189-98 Therefore, there is a need to identify 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. In addition, there is a need to identify 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.
SUMMARY
Disclosed herein are novel HLA-A3 and HLA-A2 FGF-5 epitopes, which can stimulate an immune response, and in some examples, can be used to treat an 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. As a result of these variations, HLA-A2 and HLA-A3 molecules can recognize distinct epitopes of a single protein, such as FGF-5.
Examples of particular FGF-5 HLA-A3 epitopes are disclosed herein. For example, the disclosure provides purified, immunogenic peptides which include SEQ
ID NO: 17, as well as variants thereof that can stimulate an immune response, such as SEQ ID NO: 9. In one example, such variants are recognized by, or can generate an immune cell (nominally a T-cell) that specifically reacts with SEQ ID NO: 9.
Other particular examples of 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. In one example, 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.
Antigen MHC Epitope ExpressionComments Reference Restriction on RCC
RAGE-1 B07 SPSSNRIRNT 2% Rarely on Gaugler RCC et al., 1996.
Immunogenetics 44:323-30 MN-CA 0201 HLSTAFARV 85% Difficult Vissers IX to detect et al., epitope 1999. Cancer presented on tumors* Res. 59:5554-9;
*Parkhurst, M
(personal communication) hTERT A0201 ILAKFLHWL 85% Some normalVonderheide et (teleomerase)03 KLFGVLRLK tissue expressional., 1999.
Imrnunity.
10:673-679 RU1 B51 VPYGSFKHV frequentUbiquitous Morel et on al., normal tissue2000. Immunity.
12:107-117 RU2 (AS)B07 LPRWPPPQL frequentAnti-sense Van den Eynde transcript et al., 1999.
Expressed JExp.Med.
in testis and normal 190:1793-800 kidney IntestinalB0702 SPRWWPTCL frequentAlt. ORF Ronsin epitope et al., carboxyl Expressed 1999. J
in esterase normal tissueInununol.
163:483-90 mut hsp70-20201 SLFEGIDIYT unique Tumor-specificGaudin et al., mutation 1999. J
Immurzol.
162:1730-8 M-CSF B3501 LPAWGLSPGEQEY60% Alt. ORF Probst-Kepper epitope et Expressed al., 2001.
in normal kidneyJExp.Med.
and liver 193:1189-98 Therefore, there is a need to identify 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. In addition, there is a need to identify 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.
SUMMARY
Disclosed herein are novel HLA-A3 and HLA-A2 FGF-5 epitopes, which can stimulate an immune response, and in some examples, can be used to treat an 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. As a result of these variations, HLA-A2 and HLA-A3 molecules can recognize distinct epitopes of a single protein, such as FGF-5.
Examples of particular FGF-5 HLA-A3 epitopes are disclosed herein. For example, the disclosure provides purified, immunogenic peptides which include SEQ
ID NO: 17, as well as variants thereof that can stimulate an immune response, such as SEQ ID NO: 9. In one example, such variants are recognized by, or can generate an immune cell (nominally a T-cell) that specifically reacts with SEQ ID NO: 9.
Other particular examples of 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. In one example, 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. In one example, the HLA
haplotype of the subject is determined prior to administration 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.
The foregoing and other objects, features, and advantages will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
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 D indicate internally deleted amino acid residues.
_7_ The degree of CTL recognition is categorized as follows depending on IFN-y secretion:
(++) >1000 pg/ml, (+) 200-1000 p~xnl, (+/-) 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-y 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-y assay. Error bars represent the standard deviation of duplicate IFN-y determinations.
FIG. 6 is a schematic representation of plasmids with termination codons inserted between the two determinant-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-y 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 ID 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 (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.
_g_ 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 ID NO: 1). Error bars represent the standard deviation of duplicate IFN-y determination.
FIGS. l0A-lOD 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-y determination.
FIGS. 11A and 11S are bar graphs showing that presentation of both MHC
class I-restricted peptides was blocked by clasto-Lactacystin (3-lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport. (A) Open bars:
untreated cells, filled bars: cells incubated with clasto-Lactacystin (3-lactone. (b) RCC
cell line was infected with either an adenovirus encoding GFP (open bars) or inhibitor ICP47 (filled bars). Bars represent the average IFN-y 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 L11~F amino acid substitution (A516C nucleotide substitution) in an FGF-5 HLA-AZ epitope.
SEQUENCE LISTING
The 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.
_g_ SEQ 1D 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 (YASPI2F) 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 1D NO: 11 shows amino acids 173-220 of FGF-5.
SEQ 1D 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;
Genbank Accession No: AAB06463.
SEQ 1D NO: 17 shows a variant FGF-5 HLA-A3 eptiope.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Abbreviations and Terms The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, the singular forms "a" or "an" or "the"
include plural references unless the context clearly dictates otherwise. For example, reference to "an FGF-5 epitope" includes one or a plurality of such epitopes, and reference to "the tumor" includes reference to one or more tumors and equivalents thereof known to those skilled in the art, and so forth. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. For example, the phrase "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. As used herein, "comprises" means "includes." Thus, "comprising an FGF-5 HLA-A3 epitope and IL-2," means "including an FGF-5 HLA-A3 epitope and Il-2," without excluding additional elements.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
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 subj ects.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that axe administered, 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.
Exemplary 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-diamminedichloroplatimun (II), and hydroxyurea.
Antineoplastic agents also include biologics, 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~' edition, section XIII.
Cancer: Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increase rate of growth, invasion of surrounding tissue, and is capable of metastasis.
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. In a particular example, 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 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). For example, 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. In one example, 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.
In one example, 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. In another example, two conservative substitutions are included in the peptide.
In a further example, 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. Alternatively, 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.
Examples of amino acids which may be substituted for an priginal amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg;
Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met;
Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.
Further information about conservative substitutions can be found in, among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al., (P~otein Sci. 3:240-7, 1994), Hochuli et al., (BiolTechhology 6:1321-5, 1988) and in standard textbooks of genetics and molecular biology.
Deletion: The removal of a sequence of a nucleic acid, for example I)NA, 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. In a particular example, an FGF-5 HLA-A2 or HLA-A3 epitope, or combinations thereof, enhances the benefit of 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.
In one example, an FGF-5 HLA-A2 or HLA-A3 epitope, or combinations thereof, enhances an immune response as measured by clinical response. One example of 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. For example, 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 117 NO: 7 as well as variants, fragments, and fusions thereof that retain the ability to stimulate an immune response against FGF-5. Exemplary FGF-5 HLA-A3 epitopes include, but are not limited to, SEQ 1D
NOS: 1-6, 9 and 17, as well as variants, fragments, and fusions thereof that retain the ability to stimulate an immune response against FGF-5.
Fibroblast growth factor 5 (FGF-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: M37~25 (human) and NM 010203 (mouse) and exemplary amino acid sequences include Genbank Accession Nos: AAB06463 (human) and NP 034333 (mouse). However, those skilled in the art will appreciate that other FGF-5 sequences are publicly available for several other organisms. In some examples, 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.
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.
Functionally Equivalent: The ability of a peptide containing one or more sequence alterations to retain a function of the unaltered peptide. Examples of sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, insertions, and combinations thereof.
In a particular example, an FGF-5 eptiope including one or more sequence alterations retains a function of the unaltered epitope. In one example, the variant FGF-5 eptiope specifically binds an antibody that also binds to an unaltered form of an FGF-5 eptiope. In another or additional example, 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).
In one example, a particular peptide binds an antibody, and a functional equivalent of that particular peptide is another peptide that binds the same antibody.
Thus 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). In a particular example, a functional equivalent includes a polypeptide having a discontinuous binding sequence, and the antibody binds a linear epitope. Thus, if the peptide sequence is MLSVLEIFAV (SEQ ID NO: 7) a functional equivalent includes discontinuous epitopes, which may can appear as follows (**=any number of intervening amino acids):
NH2 -**-M**L**S**V**L**E**I**F**A**V-COOH. 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, and/or 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.
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. There are five genetic loci, designated HLA-A, HLA-B, HLA-C, HLA-D, and HLA-DR. At each locus, there can be any of several different alleles.
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 minutes, 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. In one example, the response is specific for a particular antigen (an "antigen-specific response"). In one example, an immune response is a T cell response, such as a CD4+
response or a CD~+ response. In another example, the response is a B cell response, and results in the production of specific antibodies. In a particular example, 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: polymorphomnuclear 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.
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, 1 ~, 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.
Operably linked: 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. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, 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).
In one example, a peptide is at least 6 amino acids in length, such as at least ~, 9, 10, 11, or 12 amino acids in length. In particular examples, 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. In one example, 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 subj ect.
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: "Preventing" 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. Et~zymol. 153:516-44, 1987).
Specific, non-limiting examples of promoters include promoters derived from the genome of mammalian cells (such as a rnetallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; or the vaccinia virus 7.SK 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: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, 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. In another example, 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.
In one example, 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. Examples of methods that can be used to purify an antigen, include, but are not limited to the methods disclosed in Sambrook et al.
(Molecular Clorzitzg: ~. A Laboratory Manual, Cold Spring Harbor, New York, 1959, 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.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurnng 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.
Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
Specific binding agent: An agent that binds substantially only to a defined target. Thus an FGF-5 specific binding agent is an agent that binds substantially to an FGF-5 polypeptide. In one example, 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.
Methods of alignment of sequences for comparison are well known in the art.
Various programs and alignment algorithms are described in: Smith and Waterman, Adv.
Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970;
Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gehe 73:23?-244, 1988; Higgins and Sharp, CABIOS' 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.
85:2444, 1988;
and Altschul et al., Nature Genet. 6:119-129, 1994.
The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al., J. Mol.
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.
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.
For comparisons of amino acid sequences of greater than about 30 amino acids, 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). When aligning short peptides (fewer than around 30 amino acids), 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. 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 determining sequence identity over such short windows are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Maryland. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
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. In one example, a CD8 T cells is a cytotoxic T lymphocytes.
In another embodiment, 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.
Transduced and Transformed: A virus or vector "transducer" 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. As used herein, the term 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: A transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term 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. In one embodiment, a transgene encodes an FGF-5 polypeptide, such as an FGF-5 HLA-A3 or HLA-A2 epitope.
Variants or fragments or fusion proteins: The disclosed FGF-5 HLA-A3 and HLA-A2 epitopes include variants, fragments, and fusions thereof. DNA
sequences which encode for an epitope or fusion thereof, or a fragment or variant of thereof (for example a fragment or variant having at least 80%, at least 90% or at least 95%
sequence identity to an FGF-5 HLA-A3 or HLA-AZ epitope) can be engineered to allow the protein to be expressed in eukaryotic cells or organisms or bacteria. To obtain expression, 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. In one example, the other amino acid sequences are at least 8, 9, 10, 12, 15, 20, 30, or 50 amino acid residues in length.
One of ordinary skill in the art will appreciate that 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. In one example, a vector is a viral vector. Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
FGF-5 Polynucleotides and Polypeptides Previously, a renal cancer-reactive T-cell clone, Clone 2, was obtained from tumor-infiltrating lymphocytes (TILE within a RCC metastasis undergoing spontaneous regression. 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. Cancer Lett.
10:91-7; Kornmann et al., 1997. Oncogerce 15:1417-24). FGF-5 has paracrine, and possibly autocrine, trophic activity on some cultured human cancer lines and has been described to be angiogenic ih vivo in a porcine model (Giordano et al., 1996. lVat. 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.
Disclosed herein are FGF-5 HLA-A2 and HLA-A3 epitopes. In one example, 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. In a particular example, an HLA-A3 epitope is at least eight amino acids long, and includes the sequence Tyr-Ala-(A3)-(A4)-Arg-Phe wherein A3 is Ala or Ser and A4 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.
In one example, 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. In a particular example, 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. In one example, 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. Such 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. In one example, 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. In one example, 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. In a particular example, a variant includes two amino acid substitutions, or three amino acid substitutions, for example in any of SEQ m NOS: 1, 3-7, 9 and 17.
The FGF-~ HLA-AZ 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. One skilled in the art will understand that fusion proteins including an FGF-5 HLA-A2 or HLA-A3 epitope can be even longer. For example, 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-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. Also encompassed by the disclosure are 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 ID NOS: 1-7, 9, and 17, or nucleotide sequences that include such sequences) under physiological conditions. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences. 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 subj ect 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. As used herein, the term "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. Thus expression control sequences can include appropriate promoters, enhancers, transcription terniinators, 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. The term "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 minimal 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 y, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.SK promoter) can be used. 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 _29_ pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J.
Biol. Chern. 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells. 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).
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. Where 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 CaCIZ method using procedures well known in the art. Alternatively, MgCl2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinj ection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. 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 (Eukafyotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Isolation and purification of recombinantly expressed polypeptide can be performed using conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
FGF-S HLA-A2 and HLA-A3 Epitopes as Immunogenic Compositions Disclosed herein are methods for treating a subject having an FGF-5 expressing (or overexpressing) tumor. 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), of 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. In one example, 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. This allows one, such as a physician, to determine the appropriate FGF-5 epitope to administer to the subject. For example, if the subject is determined to have an HLA-A3 haplotype (that is they have at least one HLA-A3 allele), the subject can be administered an FGF-5 HLA-A3 epitope. In another example, if the subject is determined to have an HLA-A2 haplotype (that is they have at least one HLA-A2 allele), the subject can be administered an FGF-5 HLA-A2 epitope. Examples of 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.
Also disclosed are methods for eliciting an immune response in a subject. 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.
Specific, non-limiting examples of an immune response are a B cell or a T cell response. In one example, 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, such as an FGF-5 HLA-A2 or HLA-A3 antigen, are disclosed. The method includes administering to a subject a disclosed FGF-5 HLA-A2 or HLA-A3 antigen. In one example, the subject is an experimental animal, such as a mouse or rabbit. In another example, the subject is a human.
In forming a pharmaceutical composition for eliciting an immune response in a subject, or for treating an FGF-5 expressing or over-expressing tumor, one or more FGF-5 HLA-A2 or HLA-A3 epitopes (at a therapeutically effective amount), or combinations thereof, alone or in combination with other agents, is utilized.
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. For example, a variant of SEQ III NO: 7, will be recognized by an immune cell that recognizes SEQ ID NO: 7. In a particular example, such variants, fragments, and fusions, provide an advantage, such as increasing the solubility or immungenicity of the eptitope, or easing linking or coupling of the epitope.
In one example, 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. In addition, an amino acid chain corresponding to an additional antigen or immunogen can be included. Thus, an immune response to more than one antigen can be induced by immunization.
Specific non-limiting examples of 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. These 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 amino 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.
In some examples, it is desirable to combine two or more FGF-5 epitopes that contribute to stimulating specific immune responses in one or more subjects or histocompatibility types. 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). For example, 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.
In some examples, 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. When present, 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-terminus, N-terminus or a side chain of one or more of the amino acids. Examples of 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. In a particular example, butyraldehyde is used as a crosslinking agent, a divalent imido ester or a carbodiimide.
In another example, cysteine residues can be added at the amino- and carboxy termini 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. 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.
Many of these thioether forming 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. In these reagents, the carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-vitro-4-sulfonic acid, sodium salt. One coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Ideally, 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 subj ect. For example, 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.
By this means 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.
In one example, the disclosed epitopes, such as an FGF-5 HLA-A2 eptiope, for example SEQ ID NO: 7, is obtained from natural sources. In this example, an protein is subjected to selective proteolysis, for example by splitting the protein with chemical reagents or enzymes. Alternatively, because 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. Kent, Biomedical Polymers, eds. Goldberg and Nakajima, Academic Press, New York, pp. 213-242, 1980; Mitchell et al., J.
Org.
Chem., 43:2845-52, 1978; Tam et al., Tet. Letters, 4033-6, 1979; Mojsov et al., J. O~g.
Chem., 45:555-60, 1980; Tam et al., Tet. Letters, 2851-4, 1981; and Kent et al., Proceedings of the IV International Symposium on Methods of Protein Sequence Analysis, (Brookhaven Press, Brookhaven, N.Y, 1981. In addition, 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. -Determination of Therapeutic Effect Regardless of the exact formulation, 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 molecules) or compositions with binding assays, ih 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 104) or irradiated peripheral blood mononuclear cells (5 x 104) are seeded, in duplicate, into wells with or without about 200 ~,g/ml peptide (Hemmer, et al., 1998, J. l'ept. Res. 52:338-45).
Proliferation is measured by 3H-thymidine incorporation (Hemmer et al., 1997, J. Exp. Med.
185:1651-9). In one example, 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. Immuhol.
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. The 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. In one example, a therapeutic composition that includes an 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.
Diagnostic Agents The disclosed peptides can also be used as diagnostic reagents. For example, 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. As diagnostic reagents, epitopic peptides can be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual.
These and additional features are further explained by the following non-limiting examples.
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 pMEl8S and tested by transfection into HLA-A3 expressing COS7 and recognition by Clone 2 assessed as follows.
Briefly, the reactivity of 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 104 COS-A3 cells/well using Lipofectamine) in 96-well plates.
After an overnight culture, 2 x 104 cells/well of Clone 2 CTL were added. After incubating for 20-24 hours, supernatants were harvested and IFN-y concentration was analyzed by ELISA (Endogen, Woburn, MA), where IFN-y concentration is considered proportional to CTL activation. ELISA plates (96 well flat bottom, Costar, N~ were coated with anti-human IFN-y monoclonal antibody (2G1, ENDOGEN, MA) at 1 ~g/ml, 100 ~.1/well overnight. After washing, plates were blocked with PBS-5% FBS (fetal bovine serum) for one hour (200 wl/well), the samples added (50 ~,1/well) and incubated for 90 minutes. Subsequently, the plates were washed and biotin-labeled anti-human IFN-y monoclonal antibody (B 133.5, ENDOGEN, MA) was added at 0.5 wg/ml, 50 ~1/well, and incubated for one hour. 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 ~,1/well) and the optical density at 45Onm wave-length was measured. Recombinant human IFN-y (Endogen, MA) was used as a standard.
As shown in FIG. 1, 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' end of this minimal coding sequence obviated recognition by Clone 2.
HLA Class I binding peptides are typically 8-10 amino 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-y by ELISA. Stepwise internal deletions were made, starting with a recognized mini-gene to identify residues important for epitope recognition. As shown in FIG. 2, 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.
Subsequently, mini-genes with single alanine substitutions at each of the 27 codons within G41 (FIG. 3) 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 (Nl~z), 174 (Y1~4), 176 (516), and 217-220 (Pzl~, Rzia, Fzl9, and Kzzo) (with potential lesser contributions from amino acids 200 and 214). Peptides with Tyr at position 172 (position 3 of the mini-gene) and Phe/Lys at positions 219 and 220 (9/10 of the mini-gene) were favored for binding of HLA-A3 molecules.
Fusion peptides encompassing these two minimal terminal fragments were synthesized and tested for recognition by Clone 2 as follows. EBV-B cells were co incubated with listed peptide at 10 ~,M, for 30 minutes at room temperature.
After the incubation, EBV-B cells were washed and CTL were added. IFN-y in the supernatant was measured 20-24 hours later by ELISA. Peptide pulsing was done in RPMI
medium 1640 (RPMI) and the co-culture was done in RPMI with 10% fetal bovine serum (FBS).
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. 3, two peptides, a 9-mer derived from 5 N-terminal amino acids and 4 C-terminal amino acids (of 172-176 plus 217-220; SEQ ID NO: 1) is i strongly recognized, stimulating C2 cells at a concentration of 10 nM (FIG.
4). The only other stimulatory peptide was the 10-mer NTYASLPRFK (172-176 plus 216-220;
SEQ ID NO: 2), which demonstrated 1000-fold lower activity than SEQ ID NO: 1 (FIG.
5). Covalent fusion between NTYAS (amino acids 1-5 of SEQ ID NO: 1) and PRFK
(amino acids 6-9 of SEQ ID NO: 1) provided a much better result than incubating APCs with even high concentrations of these peptides separately (FIG. 4, NTYAS+PRFK) These results demonstrate that FGF-5 epitope generation involved a covalent intramolecular event involving the two terminal ends of the 172-220 peptide from FGF-5 generating the neo-epitope recognized. The determination that Clone 2 recognized 60% of RCC lines, as well as HLA-A3+ FGF-5-transfected tumor lines, demonstrated that this protein-splicing event was common in FGF-5-expressing tumors.
Titration of the 9-mer (SEQ ID NO: 1) showed it to be recognized at the nanomolar level while micromolar amounts of the 10-mer (SEQ ID NO: 2) were needed for stimulation of Clone 2. Therefore, the 9-mer neo-peptide (SEQ ID NO: 1) was processed and presented to T-cells in the context of HLA-A3 on FGF-5-expressing renal cancers.
An alanine scan was performed on the 9-mer (SEQ 1D NO: 1), using standard methods, to deternzine which amino acids could tolerate an alanine substitution, without significant loss in immunoreactivity. Position 173 of the peptide tolerates alanine substitution, and there is only partial reduction in activity with alanine substitutions at positions 172 (N1~2), 176 (516) and 217 (P21~) (see Table 2). Therefore, epitopes having one or more amino acid substitutions at positions 172 (SEQ ID NO: 4), 173 (SEQ
ID
NO: 3), 176 (SEQ ID NO: 5) or 217 (SEQ ID NO: 6) are' encompassed by this disclosure, as are other sequences with amino acid substitutions.
Table 2: Results of Alanine Scan of SEQ ID NO: 1.
Alanine SubstitutionResultin Se uence SEQ ID NO
9-mer with no substitutionNTYASPRFK SEQ ID NO: 1 (amino acids 172-176 and 217-220 of FGF-5) T173A N_AYASPRFK SEQ ID NO: 3 N172A _ATYASPRFK SEQ ID NO: 4 S 176A NTYA_APRFK SEQ ID NO: 5 P217A NTYASARFK SEQ ID NO: 6 HLA-A3 Epitope is Generated by Post-Translational Splicing This example describes the methods used to demonstrate the mechanism that generates the 9-mer fusion peptide (SEQ ID 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.
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 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.
To demonstrate that the HLA-A3 epitope (SEQ ID NO: 1) is generated by post-translational protein splicing, the processing and recognition of synthetic peptides from FGF-5 (see FIG. 8A) pulsed onto HLA-A3+ or HLA-A3- EBV transformed B (EBV-B) cells was determined as follows. EBV-B cells were co-incubated with 10 ~,M of the peptide for 3 hours at 37°C. After the incubation, EBV-B cells were washed and CTL
were added. IFN-y in the supernatant was measured 20-24 hours later by ELISA.
Peptide pulsing was done in RPMI and the co-culture was done in RPMI with 10%
FBS. Where indicated, EBV-B cells were fixed in 1% formaldehyde in PBS, for 10 minutes on ice, and washed in PBS. Peptides were synthesized and molecular weights verified as described in Example 1.
As shown in FIGS. 8B and 8C, high concentrations of a peptide corresponding to FGF-5 172-220 starting with the 5-mer (172-176, NTYAS; amino acids 1-5 of SEQ
ID NO: 1) and ending with the 4-mer (217-220, PRFK; amino acids 6-9 of SEQ ID
NO:
1) activated Clone 2 (but not another RCC-specific CTL clone) in a HLA-A3 restricted manner. Recognition was lost by deletion of residue 172 or residues 213-220 and was not affected by the addition of residues 161-171.
Presentation of the long peptide (SEQ ID NO: 13) was abrogated by mild aldehyde fixation of APCs, a process that did not affect the presentation of the 9-mer (SEQ ID NO: 1) (FIG. 9). Therefore, the results shown in 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).
To confirm this observation, synthetic or acid-stripped 9-mer (NTYASPRFK;
SEQ ID NO: 1) and 49-mer (172-220; SEQ m NO: 13) peptides were fractionated by HPLC and the capacity of fractions to activate Clone 2 determined as follows.
and COS-A3/FGF-5 were prepared by retrovirally transducing COS-T cells. Each cell line was cultured in T175 flasks and the peptides were stripped by adding 7.5 ml/flask of citrate buffer (0.13M citric acid, 0.056M sodium phosphate dibasic, pH 3.1) for 90 seconds. Peptide solutions (from 4x109 cells, 1,200 ml each) were prepared and were concentrated by Sep-pak Plus Cl8 column (Waters). After lyophilization and resolubilization into 200 ~l of 5% acetonitrile 0.05% (v/v) TFA, peptides were fractionated using a C1g 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) . Fractions (1 ml each) were collected, lyophilized, reconstituted in 100 ~1 of RPMI and added to EBV-B. After 3 hours incubation, CTL
in 100 ~,l of RPMI with 20% FBS were added and IFN-y was measured at 20 hours.
Synthetic peptides (9-mer 20 ~,g and 49-mer 100 ~,g) were HPLC fractionated and assayed in the same way as above. All the 9-mer fractions were diluted 100 times before the assay.
As shown in FIGS. l0A and l OB, the antigenic activities of the synthetic 9-mer (SEQ ID NO: 1) and 49-mer (SEQ ID NO: 13) eluted in fractions 11 and 22, respectively. These fractions contained the expected peptides as determined by mass-spectrometry. The 9-mer eluting in fraction in 11 was active using fresh or fixed APCs, while the 49-mer eluting in fraction 22 was active only using live cells.
HPLC also demonstrated that the 9-mer (SEQ ID NO: 1) represents the naturally processed peptide from FGF-5. As shown in FIG. l OD, antigenic activity of acid-s stripped peptides from COS-A3 cells expressing FGF-5 was exclusively recovered from fraction 11. By contrast, none of the fractions from non-FGF-5 expressing cells demonstrated significant antigenic activity (FIC. 1 OC). These results indicate that the naturally processed 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.
Given the unusual origin of the 9-mer determinant (SEQ ID NO: 1), the antigen processing pathway utilized to generate it from full length FGF-5 was determined. An autologous RCC cell line that expresses melanoma antigen gp100, HLA-A2, A3, and DR(31 * 0401 was generated by retroviral vector-mediated transduction of gp 100, HLA-A2, and class II transactivator CIITA genes (CIITA), (the tumor is genotypically HLA-DR(31 * 0401 ). The result is to create a tumor target capable of presenting not only its autologous antigens to Clone 2, but also class I and II-restricted determinants from gp100 to control T-cell clones which recognize this melanoma antigen (class II
gp100 recognition is restricted by DR[31 *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 p.M clasto-Lactacystin (3-Lactone (Calbiochem) for 3 hours.
After washing, T-cells were added and IFN-y was measured at 20 hours using the methods described in Example 1. TAP-1 dependency was determined by infecting the RCC
line with either an adenovirus encoding GFP or TAP-1 inhibitor ICP47 (MOI=100, 2 hours).
After washing, overnight culture and the treatment with the citric acid buffer as above, T-cells were added, and IFN-y measured at 20 hours. A CD8+ T cell clone that recognizes the gp100209-217 determinant was used as a positive control for inhibitor effectiveness and a CD4+ T cell clone that recognizes gp1OO44-59 determinant was used as a control to eliminate non-specific effects of inhibitors on general antigen presentation capacity.
As shown in FIGS. 11A and 11B, presentation ofboth MHC class I-restricted peptides was blocked by the proteasome inhibitor clasto-Lactacystin [3-lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport. MHC
class II-restricted gp100 recognition was unaffected.
Based on these results, it appears that the 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. StYUCt. Biol. 1:502-4). The proteasome and TAP-dependence of antigen presentation and the successful splicing of plasmid encoded trunctated 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.
These results also demonstrate that the immune system monitors non-contiguous peptide sequences generated post-translationally. This capability represents an enormous increase in the ability of CTLs to recognize self and foreign proteins. While this could enhance immune surveillance, it does complicate the task of identifying peptide ligands recognized by tumor- and pathogen-specific CTL.
Identification of an HLA-A2-Restricted Epitope To broaden the subject base that could participate in vaccine trials targeting FGF-5, an HLA-A2-restricted epitope of FGF-5 was identified using the following methods.
Peripheral blood lymphocytes (PBL) from HLA-A2 subj ects 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.
Collins, CO), that was reportedly a peptide from the N-terminal leader sequence of FGF-5, and repetitively stimulating PBL from an HLA-A2 positive RCC subject using the synthetic peptide preparation containing FGF-5 sequences, a culture which recognized the stimulating peptide preparation pulsed onto A2+ cells (but not a control peptide) and recognized an FGF-5 expressing HLA-A2+ RCC line (but not the same tumor without HLA-A2) was obtained. This was cloned by limiting dilution and six clones with the same reactivity were obtained. These clones recognized peptide, FGF-5+/HLA-A2+ tumor as well as HLA-A2+ COS cells transfected with the gene for FGF-5, but not the control vector, confirming that the peptide epitope was naturally processed from full length FGF-5 and presented on HLA-A2 (FIG. 12).
Unfortunately, mass spectroscopy of preparation 5194 demonstrated that it was not pure, and instead contained a number of peptides. The actual peptide recognized was determined by testing truncations of the FGF-5 gene transfected into HLA-A2+
cells. A2+ target cells transfected with the full-length FGF-5 gene were recognized.
Truncations of the FGF-5 gene localized the epitope to a region overlapping nucleotides 467-541 of FGF-5 (Genbank Accession No: M37825). A candidate A2-binding 10-rner 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).
To further demonstrate that 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 (L118F) at position two of SEQ ID 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.
Overview of Methods of Using FGF-5 Epitopes Having demonstrated that tumor-reactive T-cells generated from subjects with RCC can recognize naturally presented FGF-5 in either the context of HLA-A2 or HLA-A3 via the minimal determinants MLSVLEIFAV (SEQ ID NO: 7) or NTYASPRFK (SEQ ID NO: 1), respectively, such 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.
' Disclosed in the examples below are methods that one skilled in the art can use to determine response rates and toxicity of peptide vaccination with SEQ ID
NO: 1 or 7 (or variants or fusions thereof) in HLA-appropriate subjects with RCC and determine the effect of such administration on the response rate to high-dose IL-2. This is done as vaccine-only in subjects who are not candidates for IL-2 therapy or with IL-2 therapy in subjects who are eligible to receive 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 (IFA) 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. They receive vaccination and high-dose bolus IL-2 along with peptide (in the presence of IFA) vaccine with clinical response and immunological response to vaccination as primary endpoints.
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.
Administration of FGF-5 Peptides 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. In this example, 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 A begin receiving vaccination with HLA-appropriate peptide emulsified in IFA every 3 weeks. This is continued for up to a year, or until tumor progression (see Example 9) is documented. At that point, those eligible for IL-2 who have not yet received it, can have high-dose intravenous bolus IL-2 added to their peptide vaccination regimen. Throughout, all IL-2 is administered at 720,000 IU/kg/dose every 8 hours by intravenous bolus to the maximum tolerated number of doses (which constitutes one cycle of therapy). Two cycles, separated by 10-14 days, is administered during every two month period, with further treatment dependent on interval tumor assessment. For subjects in Cohort A crossing over to vaccination plus IL-2 therapy, a single peptide + IFA vaccination is administered the day prior to starting an IL-2 cycle (instead of every three weeks, to accommodate the standardized regimen).
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.
Subjects with advanced loco-regional disease have a poor prognosis and no adjuvant therapy options of efficacy. Therefore, Cohort C includes subjects who have undergone resection of either T3/T4 or Nl/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.
Within each cohort, 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 ID
NO: l, 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 (SEQ ID NO: 1) in IFA. The 1 mg dose of peptide was selected based on immunization with peptides derived from the MART-1 and gp100 melanoma antigens, where no significant dose response relationship was observed over 0.1 mg to 10 mg of peptide (Cormier et al., 1997. Cancer J. Sci. Am. 3:37-44; Salgaller et al., 1996 Cancer Res.
56:4749-57).
Table 3: Examples of Treatment Methods (Each cohort has subgroups for HLA-A2 and HLA-A3) Cohort A Cohort B Cohort C
Metastatic disease Metastatic disease - Resected Stage III
disease able to delay IL-2 receives immediate Starts vaccine aloneStarts standard Starts vaccine once IL-2 with alone every 3wks vaccine rior to once every 3wks each cycle If PD seen, and If PD seen, patientIf relapse seen, IL2- taken without eligible, start off of protocol surgical option, standard IL- starts 2 with vaccine prior standard IL-2 to with each cycle vaccine prior to each cycle Evaluations once once every 2 monthsonce every 3 months every 6 while weeks on peptide on IL-2; once everywhile on peptide and once 3-6 once every 3-6 months months if stable every 2 months if stable after while beyond 6 months; , on IL-2; once once every 3-6 every 2 months while months if stable on after Feptides 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 Systems (San Diego, CA). 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; O.1N HCl 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
675756) and sterile water for injection in the anterior thigh deep subcutaneous tissue as follows. To prepare a 1 mg dose, add 1.5 ml of Montanide ISA-51 to a vial of the peptide (1 mg/ml, 1.5 ml ml/vial). The vial is vortexed for 12 minutes, and 2.0 ml is withdrawn for administration.
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.
Other adjuvants can be used, for example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
Ihterleuki~a-2: Iht~avenous Administration In particular examples, IL-2 (Chiron Corp., Emeryville, CA) is administered at a dose of 720,000 ICT/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 rnl 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 xnl of 5% human serum albumin (HSA).
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 mp~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.
Evaluation During Treatment In a particular example, 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 administered as an outpatient, unless inpatient admission is indicated for treatment of vaccine-related side-effects or underlying disease management.
Subjects are monitored after each injection with vital signs twice within the first hour and are discharged from clinic at that time if stable. 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.
Subject peripheral blood lymphocytes (PBL) can be purified by centrifugation on a Ficoll cushion, then incubated in the presence of the vaccinating peptide, and separately in the presence of a control peptide, to stimulate proliferation of specific CTL precursors, as previously described (Cormier et al., 1997. Cancer J. Sci.
Am. 3:37-44; Salgaller et al., 1996 Cancer Res. 56:4749-57). 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.
Evaluation Following Treatment For subjects in cohort A on peptide vaccine alone, complete physical evaluation, CBC, acute care, hepatic and mineral panels and appropriate X-ray evaluations of all evaluable lesions are obtained every six weeks during the first six months of therapy and if stable, every 3-6 months thereafter. Other evaluations can be performed as indicated by symptoms or physical findings.
For cohorts A and B during peptide vaccine plus high-dose IL-2 therapy, complete physical evaluation, CBC, acute care, hepatic and mineral panels and appropriate X-ray evaluations of all evaluable lesions are obtained every two months while on IL-2, and every 3-6 months for stable subjects off therapy.
For cohort C, complete physical evaluation, CBC, acute care, hepatic and mineral panels are performed every three months for the first year and every six months thereafter. Surveillance CT of the chest, abdomen and pelvis are obtained with at least every other assessment and as indicated by symptoms or physical findings.
Additional Treatment In particular examples, if 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.
Evaluation of Responsive Criteria Evaluatioia of target lesiofas All 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 fiu~iher characterize the objective tumor response of the measurable dimension of the disease as outlined in Table 4.
Table 4: Measurement of Target Lesion Response.
Complete Response Disappearance of all target lesions (CR) Partial Response At least a 30% decrease in the sum (PR) of the longest diameter (LD) of target lesions taking as reference the baseline sum LD.
Progression (PD) At least a 20% increase in the sum of LD of target lesions taking as reference the smallest sum LD
recorded since the treatment started or the appearance of one or more new lesions.
Stable Disease Neither sufficient shrinkage to qualify (SD) for PR nor sufficient increase to qualify for PD taking as references the smallest sum LD.
Mixed Res onse A shrinka a of some lesions but an increase in others Evaluation of uou-target lesions All other lesions (or sites of disease) are identified as non-target lesions and recorded at baseline. Measurements are not required, and these lesions are followed as "present" or "absent."
Table 5: Measurement of Non-Target Lesion Response.
Complete Response Disappearance of all non-target (CR): lesions and normalization of tumor marker level.
Non-Com lete Res Persistence of one or more non-target onse: lesions Progression (PD): Appearance of one or more new lesions.
Unequivocal progression of existing non-target lesions Evaluation of best ~verall respofzse The best overall response is the best response recorded from the start of the treatment until disease progressionlrecurrence (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.
Table 6: Evaluation of Overall Response.
Tar et Lesions Non-Tar et LesionsNew Lesions Overall Res onse CR CR No CR
CR Non-CR/Non-PD No PR
PR Non-PD No PR
SD Non-PD No SD
PD Any Yes or No PD
Any PD Yes or No PD
Any An Yes PD
Coufirmatory MeasurerzzeutlDuration of Response Confirmation To be assigned a status of PR or CR, changes in tumor measurements are confirmed by repeat studies that are performed at least 4 weeks after the criteria for response are first met. ~ In the case of SD, follow-up measurements met the SD
criteria at least once after study entry at a minimum interval of 6-8 weeks.
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.
Duration of stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started.
Administration of FGF-5 HLA-A2 and HLA-A3 Epitopes 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+. However, one skilled in the art will understand that other 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. In addition to administering only an FGF-5 HLA-A3 or -A2 epitope to a subject, 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+
For example, using the methods described in Example 5, a combination of FGF-5 HLA-A3 and -A2 epitopes are administered to a subject at a therapeutically effective dose. In a particular example, 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-peptide (such as SEQ ID NO: 1, 9, 17, or a variant, fragment, or fusion thereof) in IFA.
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 -peptides, the FGF-5 expressing or overexpressing tumor can be monitored for regression.
Administering the FGF-5 HLA-AZ and HLA-A3 epitopes of the present disclosure can be accomplished by any means known to the skilled artisan. For example, a pharmaceutically acceptable carrier can be provided for the FGF-5 and HLA-A3 epitopes disclosed herein. However, a pharmaceutically acceptable carrier may not be required to induce an immune response to the FGF-5 HLA-A2 or HLA-A3 epitope. Examples of 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. Thus, a wide variety of pharmaceutically acceptable Garners 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. If 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. In one example, 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. In other words, 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. Alternatively, 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 (C2 -Clo) 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. Usually, 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.
In one example, FGF-5 HLA-A2 or HLA-A3 eptitopic peptides (or combinations thereof) 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 i~ 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, Treyads 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.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
Illustrative phospholipids include egg phosphatidylcholine, dipalinitoylphosphatidylcholine 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.
In the case of a liposomal targeted delivery system, 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.
In one example, a liposome includes the desired peptide and is directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic epitope. A variety of methods are available for preparing liposomes and are described, for example, in Szoka et al., 1980, Ann. Rev. Biophys. Bioeng 9:467 and U.S.
Pat. Nos.
4,235,871, 4,501,728, 4,837,028, and 5,019,369. 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. For example, 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.
For administration of nucleic acid molecules, 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. In one example, the retroviral vector is a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney marine leukemia virus (MoMuLV), Harvey marine sarcoma virus (HaMuSV), marine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). When the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) can be utilized. A
number of additional 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. By inserting 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. Preferred targeting is accomplished by using 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.
Since recombinant retroviruses are defective, they need assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using 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.
The pharmaceutical 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. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-33, 1990 (incorporated herein by reference).
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. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
Various considerations are described, e.g., in Gilinan et al., eds., Goodman and Gilman:
Tlae Pharmacological Bases of Therapeutics, ~th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17~' ed., Mack Publishing Co., Easton, Pa., 1990. Typically, 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. In one example, the dose is about 1.0 p,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. In one example, 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. In the case of a more aggressive disease 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.
In one example, purified therapeutically active molecules are combined with 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. In general, the nature of the pharmaceutically acceptable carrier will depend on the particular mode of administration being employed. For instance, 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. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, 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.
Pharmaceutical preparations 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.
As is known in the art, protein-based pharmaceuticals may be only inefficiently delivered through ingestion. However, 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 administering 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.
For oral administration, 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%.
For aerosol adnvnistration, 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. Mixed esters, such as mixed or natural glycerides may also be employed.
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.
Peptide Modifications 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. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1RZ wherein Rl and RZ are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, malefic, tartaric and other organic salts, or may be modified to Cl-C16 allcyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide side chains may be converted to C1-Cls alkoxy or to a Cl-C16 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 C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and,esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the disclosed peptides to select and provide conformational constraints to the structure that result in enhanced stability.
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. For computer modeling applications, 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). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press:
Buffalo Grove, IL, pp. 165-174 and Py~inciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CARD. Also included within the scope of the disclosure are mimetics prepared using such techniques. In one example, a mimetic mimics the immune response generated by an FGF-5 HLA-A3 or HLA-A2 epitope.
Determination of a Therapeutically Effective Amount This example describes methods that can be used to identify different therapeutically effective doses of an FGF-5 HLA-A3 epitope, FGF-5 HLA-A2 epitope, or combinations thereof. In one example, 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.
One example of 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.
In another or additional example, it is an amount sufficient to increase the efficacy of another agent, such as IL-2. In one example, 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.
An effective amount of FGF-5 HLA-A3 or HLA-A2 epitope protein, autologous CTLs specific to FGF-5, or combinations thereof, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, 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 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. For example, 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 methods disclosed herein have equal application in medical and veterinary settings. Therefore, 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.
In view of the many possible embodiments to which the principles of our disclosure may be applied, it is be recognized that the illustrated embodiments are only examples of the disclosure and are not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
SEQUENCE LISTING
<110> The Government of the United States of America <120> IMMUNOGENIC EPITOPES FOR FIBROBLAST GROWTH FACTOR 5 (FGF-5) <130> 67015 <150> US 60/427,920 <151> 2002-11-19 <160> 17 <170> PatentIn version 3.2 <210> 1 <211> 9 <212> PRT
<213> homo sapiens <400> 1 Asn Thr Tyr Ala Ser Pro Arg Phe Lys <210> 2 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Artifical peptide sequence.
<400> 2 Asn Thr Tyr Ala Ser Leu Pro Arg Phe Lys <210> 3 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 3 Asn Ala Tyr Ala Ser Pro Arg Phe Lys <210> 4 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 4 Ala Thr Tyr Ala Ser Pro Arg Phe Lys <210> 5 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 5 Asn Thr Tyr Ala Ala Pro Arg Phe Lys <210> 6 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 6 Asn Thr Tyr Ala Ser Ala Arg Phe Lys <210> 7 <211> 10 <212> PRT
<213> homo Sapiens <400> 7 Met Leu Ser Val Leu Glu Ile Phe Ala Val <210> 8 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 8 Met Phe Ser Val Leu Glu Ile Phe Ala Val <210> 9 <211> 6 <212> PRT
<213> homo Sapiens <400> 9 Tyr Ala Ser Pro Arg Phe <210> 10 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 10 Asn Thr Tyr Phe Leu Pro Arg Phe Lys <210> 11 <211> 48 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 11 Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys <210> 12 <211> 52 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 12 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser <210> 13 <211> 49 <212> PRT
<213> Artificial Sequence <220> .
<223> Variant peptide sequence.
<400> 13 Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe 35 40 45 , Lys <210> 14 <211> 60 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 14 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys <210> 15 <211> 1123 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (140)..(943) <400> 15 cctctcccct tctcttcccc gaggctatgt ccacccggtg cggcgaggcg ggcagagcca 60 gaggcacgca gccgcacagg ggctacagag cccagaatca gccctacaag atgcacttag 120 gacccccgcg gctggaaga atg agc ttg tcc ttc ctc ctc ctc ctc ttc ttc 172 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe agc cac ctg atc ctc agc gcc tgg get cac ggg gag aag cgt ctc gcc 220 Ser His Leu Ile Leu Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala ccc aaa ggg caa ccc gga ccc get gcc act gat agg aac cct ata ggc 268 Pro Lys Gly Gln Pro Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly tcc agc agc aga cag agc agc agt agc get atg tct tcc tct tct gcc 316 Ser Ser Ser Arg Gln Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala tcc tcc tcc ccc gca get tct ctg ggc agc caa gga agt ggc ttg gag 364 Ser Ser Ser Pro Ala Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu cag agc agt ttc cag tgg agc ccc tcg ggg cgc cgg acc ggc agc ctc 412 Gln Ser Ser Phe Gln Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu tac tgc aga gtg ggc atc ggt ttc cat ctg cag atc tac ccg gat ggc 460 Tyr Cys Arg Val Gly Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly aaa gtc aat gga tcc cac gaa gcc aat atg tta agt gtt ttg gaa ata 508 Lys Val Asn Gly Ser His Glu Ala Asn Met Leu Ser Val Leu Glu Ile ttt get gtg tct cag ggg att gta gga ata cga gga gtt ttc agc aac 556 Phe Ala Val Ser Gln Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn aaa ttt tta gcg atg tca aaa aaa gga aaa ctc cat gca agt gcc aag 604 Lys Phe Leu Ala Met Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys ttc aca gat gac tgc aag ttc agg gag cgt ttt caa gaa aat agc tat 652 Phe Thr Asp Asp Cys Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr aat acc tat gcc tca gca ata cat aga act gaa aaa aca ggg cgg gag 700 Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu tgg tat gtt gcc ctg aat aaa aga gga aaa gcc aaa cga ggg tgc agc 748 Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser ccc cgg gtt aaa ccc cag cat atc tct acc cat ttt ctt cca aga ttc 796 Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe aag cag tcg gag cag cca gaa ctt tct ttc acg gtt act gtt cct gaa 844 Lys Gln Ser Glu Gln Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu aag aaa aat cca cct agc cct atc aag tca aag att ccc ctt tct gca 892 Lys Lys Asn Pro Pro Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala cct cgg aaa aat acc aac tca gtg aaa tac aga ctc aag ttt cgc ttt 940 Pro Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe gga taatattaat cttggccttg tgagaaacca ttctttcccc tcaggagttt 993 Gly ctataggtgt cttcagagtt ctgaagaaaa attactggac acagcttcag ctatacttac 1053 actgtattga agtcacgtca tttgtttcag tgtgactgaa acaaaatgtt ttttgatagg 1113 aaggaaactg 1123 <210> 16 <211> 268 <212> PRT
<213> Homo Sapiens <400> 16 Met Ser Leu Ser'Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu r Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly <210> 17 <211> 6 <212> PRT
<213> Artificial <220>
<223> Artificial peptide.
<220>
<221> MISC FEATURE
<222> (3) . . (3) <223> Xaa is Ala or Ser <220>
<221> MISC_FEATURE
<222> (4). (4) <223> Xaa is Ala or Pro <400> 17 Tyr Ala Xaa Xaa Arg Phe
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. In one example, the HLA
haplotype of the subject is determined prior to administration 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.
The foregoing and other objects, features, and advantages will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
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 D indicate internally deleted amino acid residues.
_7_ The degree of CTL recognition is categorized as follows depending on IFN-y secretion:
(++) >1000 pg/ml, (+) 200-1000 p~xnl, (+/-) 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-y 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-y assay. Error bars represent the standard deviation of duplicate IFN-y determinations.
FIG. 6 is a schematic representation of plasmids with termination codons inserted between the two determinant-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-y 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 ID 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 (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.
_g_ 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 ID NO: 1). Error bars represent the standard deviation of duplicate IFN-y determination.
FIGS. l0A-lOD 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-y determination.
FIGS. 11A and 11S are bar graphs showing that presentation of both MHC
class I-restricted peptides was blocked by clasto-Lactacystin (3-lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport. (A) Open bars:
untreated cells, filled bars: cells incubated with clasto-Lactacystin (3-lactone. (b) RCC
cell line was infected with either an adenovirus encoding GFP (open bars) or inhibitor ICP47 (filled bars). Bars represent the average IFN-y 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 L11~F amino acid substitution (A516C nucleotide substitution) in an FGF-5 HLA-AZ epitope.
SEQUENCE LISTING
The 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.
_g_ SEQ 1D 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 (YASPI2F) 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 1D NO: 11 shows amino acids 173-220 of FGF-5.
SEQ 1D 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;
Genbank Accession No: AAB06463.
SEQ 1D NO: 17 shows a variant FGF-5 HLA-A3 eptiope.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Abbreviations and Terms The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, the singular forms "a" or "an" or "the"
include plural references unless the context clearly dictates otherwise. For example, reference to "an FGF-5 epitope" includes one or a plurality of such epitopes, and reference to "the tumor" includes reference to one or more tumors and equivalents thereof known to those skilled in the art, and so forth. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. For example, the phrase "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. As used herein, "comprises" means "includes." Thus, "comprising an FGF-5 HLA-A3 epitope and IL-2," means "including an FGF-5 HLA-A3 epitope and Il-2," without excluding additional elements.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
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 subj ects.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that axe administered, 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.
Exemplary 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-diamminedichloroplatimun (II), and hydroxyurea.
Antineoplastic agents also include biologics, 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~' edition, section XIII.
Cancer: Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increase rate of growth, invasion of surrounding tissue, and is capable of metastasis.
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. In a particular example, 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 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). For example, 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. In one example, 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.
In one example, 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. In another example, two conservative substitutions are included in the peptide.
In a further example, 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. Alternatively, 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.
Examples of amino acids which may be substituted for an priginal amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg;
Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met;
Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.
Further information about conservative substitutions can be found in, among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al., (P~otein Sci. 3:240-7, 1994), Hochuli et al., (BiolTechhology 6:1321-5, 1988) and in standard textbooks of genetics and molecular biology.
Deletion: The removal of a sequence of a nucleic acid, for example I)NA, 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. In a particular example, an FGF-5 HLA-A2 or HLA-A3 epitope, or combinations thereof, enhances the benefit of 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.
In one example, an FGF-5 HLA-A2 or HLA-A3 epitope, or combinations thereof, enhances an immune response as measured by clinical response. One example of 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. For example, 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 117 NO: 7 as well as variants, fragments, and fusions thereof that retain the ability to stimulate an immune response against FGF-5. Exemplary FGF-5 HLA-A3 epitopes include, but are not limited to, SEQ 1D
NOS: 1-6, 9 and 17, as well as variants, fragments, and fusions thereof that retain the ability to stimulate an immune response against FGF-5.
Fibroblast growth factor 5 (FGF-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: M37~25 (human) and NM 010203 (mouse) and exemplary amino acid sequences include Genbank Accession Nos: AAB06463 (human) and NP 034333 (mouse). However, those skilled in the art will appreciate that other FGF-5 sequences are publicly available for several other organisms. In some examples, 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.
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.
Functionally Equivalent: The ability of a peptide containing one or more sequence alterations to retain a function of the unaltered peptide. Examples of sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, insertions, and combinations thereof.
In a particular example, an FGF-5 eptiope including one or more sequence alterations retains a function of the unaltered epitope. In one example, the variant FGF-5 eptiope specifically binds an antibody that also binds to an unaltered form of an FGF-5 eptiope. In another or additional example, 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).
In one example, a particular peptide binds an antibody, and a functional equivalent of that particular peptide is another peptide that binds the same antibody.
Thus 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). In a particular example, a functional equivalent includes a polypeptide having a discontinuous binding sequence, and the antibody binds a linear epitope. Thus, if the peptide sequence is MLSVLEIFAV (SEQ ID NO: 7) a functional equivalent includes discontinuous epitopes, which may can appear as follows (**=any number of intervening amino acids):
NH2 -**-M**L**S**V**L**E**I**F**A**V-COOH. 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, and/or 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.
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. There are five genetic loci, designated HLA-A, HLA-B, HLA-C, HLA-D, and HLA-DR. At each locus, there can be any of several different alleles.
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 minutes, 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. In one example, the response is specific for a particular antigen (an "antigen-specific response"). In one example, an immune response is a T cell response, such as a CD4+
response or a CD~+ response. In another example, the response is a B cell response, and results in the production of specific antibodies. In a particular example, 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: polymorphomnuclear 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.
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, 1 ~, 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.
Operably linked: 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. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, 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).
In one example, a peptide is at least 6 amino acids in length, such as at least ~, 9, 10, 11, or 12 amino acids in length. In particular examples, 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. In one example, 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 subj ect.
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: "Preventing" 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. Et~zymol. 153:516-44, 1987).
Specific, non-limiting examples of promoters include promoters derived from the genome of mammalian cells (such as a rnetallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; or the vaccinia virus 7.SK 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: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, 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. In another example, 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.
In one example, 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. Examples of methods that can be used to purify an antigen, include, but are not limited to the methods disclosed in Sambrook et al.
(Molecular Clorzitzg: ~. A Laboratory Manual, Cold Spring Harbor, New York, 1959, 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.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurnng 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.
Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
Specific binding agent: An agent that binds substantially only to a defined target. Thus an FGF-5 specific binding agent is an agent that binds substantially to an FGF-5 polypeptide. In one example, 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.
Methods of alignment of sequences for comparison are well known in the art.
Various programs and alignment algorithms are described in: Smith and Waterman, Adv.
Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970;
Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gehe 73:23?-244, 1988; Higgins and Sharp, CABIOS' 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.
85:2444, 1988;
and Altschul et al., Nature Genet. 6:119-129, 1994.
The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al., J. Mol.
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.
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.
For comparisons of amino acid sequences of greater than about 30 amino acids, 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). When aligning short peptides (fewer than around 30 amino acids), 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. 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 determining sequence identity over such short windows are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Maryland. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
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. In one example, a CD8 T cells is a cytotoxic T lymphocytes.
In another embodiment, 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.
Transduced and Transformed: A virus or vector "transducer" 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. As used herein, the term 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: A transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term 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. In one embodiment, a transgene encodes an FGF-5 polypeptide, such as an FGF-5 HLA-A3 or HLA-A2 epitope.
Variants or fragments or fusion proteins: The disclosed FGF-5 HLA-A3 and HLA-A2 epitopes include variants, fragments, and fusions thereof. DNA
sequences which encode for an epitope or fusion thereof, or a fragment or variant of thereof (for example a fragment or variant having at least 80%, at least 90% or at least 95%
sequence identity to an FGF-5 HLA-A3 or HLA-AZ epitope) can be engineered to allow the protein to be expressed in eukaryotic cells or organisms or bacteria. To obtain expression, 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. In one example, the other amino acid sequences are at least 8, 9, 10, 12, 15, 20, 30, or 50 amino acid residues in length.
One of ordinary skill in the art will appreciate that 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. In one example, a vector is a viral vector. Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
FGF-5 Polynucleotides and Polypeptides Previously, a renal cancer-reactive T-cell clone, Clone 2, was obtained from tumor-infiltrating lymphocytes (TILE within a RCC metastasis undergoing spontaneous regression. 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. Cancer Lett.
10:91-7; Kornmann et al., 1997. Oncogerce 15:1417-24). FGF-5 has paracrine, and possibly autocrine, trophic activity on some cultured human cancer lines and has been described to be angiogenic ih vivo in a porcine model (Giordano et al., 1996. lVat. 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.
Disclosed herein are FGF-5 HLA-A2 and HLA-A3 epitopes. In one example, 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. In a particular example, an HLA-A3 epitope is at least eight amino acids long, and includes the sequence Tyr-Ala-(A3)-(A4)-Arg-Phe wherein A3 is Ala or Ser and A4 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.
In one example, 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. In a particular example, 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. In one example, 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. Such 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. In one example, 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. In one example, 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. In a particular example, a variant includes two amino acid substitutions, or three amino acid substitutions, for example in any of SEQ m NOS: 1, 3-7, 9 and 17.
The FGF-~ HLA-AZ 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. One skilled in the art will understand that fusion proteins including an FGF-5 HLA-A2 or HLA-A3 epitope can be even longer. For example, 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-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. Also encompassed by the disclosure are 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 ID NOS: 1-7, 9, and 17, or nucleotide sequences that include such sequences) under physiological conditions. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences. 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 subj ect 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. As used herein, the term "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. Thus expression control sequences can include appropriate promoters, enhancers, transcription terniinators, 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. The term "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 minimal 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 y, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.SK promoter) can be used. 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 _29_ pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J.
Biol. Chern. 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells. 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).
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. Where 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 CaCIZ method using procedures well known in the art. Alternatively, MgCl2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinj ection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. 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 (Eukafyotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Isolation and purification of recombinantly expressed polypeptide can be performed using conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
FGF-S HLA-A2 and HLA-A3 Epitopes as Immunogenic Compositions Disclosed herein are methods for treating a subject having an FGF-5 expressing (or overexpressing) tumor. 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), of 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. In one example, 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. This allows one, such as a physician, to determine the appropriate FGF-5 epitope to administer to the subject. For example, if the subject is determined to have an HLA-A3 haplotype (that is they have at least one HLA-A3 allele), the subject can be administered an FGF-5 HLA-A3 epitope. In another example, if the subject is determined to have an HLA-A2 haplotype (that is they have at least one HLA-A2 allele), the subject can be administered an FGF-5 HLA-A2 epitope. Examples of 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.
Also disclosed are methods for eliciting an immune response in a subject. 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.
Specific, non-limiting examples of an immune response are a B cell or a T cell response. In one example, 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, such as an FGF-5 HLA-A2 or HLA-A3 antigen, are disclosed. The method includes administering to a subject a disclosed FGF-5 HLA-A2 or HLA-A3 antigen. In one example, the subject is an experimental animal, such as a mouse or rabbit. In another example, the subject is a human.
In forming a pharmaceutical composition for eliciting an immune response in a subject, or for treating an FGF-5 expressing or over-expressing tumor, one or more FGF-5 HLA-A2 or HLA-A3 epitopes (at a therapeutically effective amount), or combinations thereof, alone or in combination with other agents, is utilized.
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. For example, a variant of SEQ III NO: 7, will be recognized by an immune cell that recognizes SEQ ID NO: 7. In a particular example, such variants, fragments, and fusions, provide an advantage, such as increasing the solubility or immungenicity of the eptitope, or easing linking or coupling of the epitope.
In one example, 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. In addition, an amino acid chain corresponding to an additional antigen or immunogen can be included. Thus, an immune response to more than one antigen can be induced by immunization.
Specific non-limiting examples of 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. These 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 amino 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.
In some examples, it is desirable to combine two or more FGF-5 epitopes that contribute to stimulating specific immune responses in one or more subjects or histocompatibility types. 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). For example, 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.
In some examples, 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. When present, 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-terminus, N-terminus or a side chain of one or more of the amino acids. Examples of 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. In a particular example, butyraldehyde is used as a crosslinking agent, a divalent imido ester or a carbodiimide.
In another example, cysteine residues can be added at the amino- and carboxy termini 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. 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.
Many of these thioether forming 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. In these reagents, the carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-vitro-4-sulfonic acid, sodium salt. One coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Ideally, 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 subj ect. For example, 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.
By this means 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.
In one example, the disclosed epitopes, such as an FGF-5 HLA-A2 eptiope, for example SEQ ID NO: 7, is obtained from natural sources. In this example, an protein is subjected to selective proteolysis, for example by splitting the protein with chemical reagents or enzymes. Alternatively, because 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. Kent, Biomedical Polymers, eds. Goldberg and Nakajima, Academic Press, New York, pp. 213-242, 1980; Mitchell et al., J.
Org.
Chem., 43:2845-52, 1978; Tam et al., Tet. Letters, 4033-6, 1979; Mojsov et al., J. O~g.
Chem., 45:555-60, 1980; Tam et al., Tet. Letters, 2851-4, 1981; and Kent et al., Proceedings of the IV International Symposium on Methods of Protein Sequence Analysis, (Brookhaven Press, Brookhaven, N.Y, 1981. In addition, 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. -Determination of Therapeutic Effect Regardless of the exact formulation, 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 molecules) or compositions with binding assays, ih 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 104) or irradiated peripheral blood mononuclear cells (5 x 104) are seeded, in duplicate, into wells with or without about 200 ~,g/ml peptide (Hemmer, et al., 1998, J. l'ept. Res. 52:338-45).
Proliferation is measured by 3H-thymidine incorporation (Hemmer et al., 1997, J. Exp. Med.
185:1651-9). In one example, 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. Immuhol.
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. The 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. In one example, a therapeutic composition that includes an 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.
Diagnostic Agents The disclosed peptides can also be used as diagnostic reagents. For example, 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. As diagnostic reagents, epitopic peptides can be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual.
These and additional features are further explained by the following non-limiting examples.
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 pMEl8S and tested by transfection into HLA-A3 expressing COS7 and recognition by Clone 2 assessed as follows.
Briefly, the reactivity of 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 104 COS-A3 cells/well using Lipofectamine) in 96-well plates.
After an overnight culture, 2 x 104 cells/well of Clone 2 CTL were added. After incubating for 20-24 hours, supernatants were harvested and IFN-y concentration was analyzed by ELISA (Endogen, Woburn, MA), where IFN-y concentration is considered proportional to CTL activation. ELISA plates (96 well flat bottom, Costar, N~ were coated with anti-human IFN-y monoclonal antibody (2G1, ENDOGEN, MA) at 1 ~g/ml, 100 ~.1/well overnight. After washing, plates were blocked with PBS-5% FBS (fetal bovine serum) for one hour (200 wl/well), the samples added (50 ~,1/well) and incubated for 90 minutes. Subsequently, the plates were washed and biotin-labeled anti-human IFN-y monoclonal antibody (B 133.5, ENDOGEN, MA) was added at 0.5 wg/ml, 50 ~1/well, and incubated for one hour. 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 ~,1/well) and the optical density at 45Onm wave-length was measured. Recombinant human IFN-y (Endogen, MA) was used as a standard.
As shown in FIG. 1, 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' end of this minimal coding sequence obviated recognition by Clone 2.
HLA Class I binding peptides are typically 8-10 amino 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-y by ELISA. Stepwise internal deletions were made, starting with a recognized mini-gene to identify residues important for epitope recognition. As shown in FIG. 2, 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.
Subsequently, mini-genes with single alanine substitutions at each of the 27 codons within G41 (FIG. 3) 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 (Nl~z), 174 (Y1~4), 176 (516), and 217-220 (Pzl~, Rzia, Fzl9, and Kzzo) (with potential lesser contributions from amino acids 200 and 214). Peptides with Tyr at position 172 (position 3 of the mini-gene) and Phe/Lys at positions 219 and 220 (9/10 of the mini-gene) were favored for binding of HLA-A3 molecules.
Fusion peptides encompassing these two minimal terminal fragments were synthesized and tested for recognition by Clone 2 as follows. EBV-B cells were co incubated with listed peptide at 10 ~,M, for 30 minutes at room temperature.
After the incubation, EBV-B cells were washed and CTL were added. IFN-y in the supernatant was measured 20-24 hours later by ELISA. Peptide pulsing was done in RPMI
medium 1640 (RPMI) and the co-culture was done in RPMI with 10% fetal bovine serum (FBS).
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. 3, two peptides, a 9-mer derived from 5 N-terminal amino acids and 4 C-terminal amino acids (of 172-176 plus 217-220; SEQ ID NO: 1) is i strongly recognized, stimulating C2 cells at a concentration of 10 nM (FIG.
4). The only other stimulatory peptide was the 10-mer NTYASLPRFK (172-176 plus 216-220;
SEQ ID NO: 2), which demonstrated 1000-fold lower activity than SEQ ID NO: 1 (FIG.
5). Covalent fusion between NTYAS (amino acids 1-5 of SEQ ID NO: 1) and PRFK
(amino acids 6-9 of SEQ ID NO: 1) provided a much better result than incubating APCs with even high concentrations of these peptides separately (FIG. 4, NTYAS+PRFK) These results demonstrate that FGF-5 epitope generation involved a covalent intramolecular event involving the two terminal ends of the 172-220 peptide from FGF-5 generating the neo-epitope recognized. The determination that Clone 2 recognized 60% of RCC lines, as well as HLA-A3+ FGF-5-transfected tumor lines, demonstrated that this protein-splicing event was common in FGF-5-expressing tumors.
Titration of the 9-mer (SEQ ID NO: 1) showed it to be recognized at the nanomolar level while micromolar amounts of the 10-mer (SEQ ID NO: 2) were needed for stimulation of Clone 2. Therefore, the 9-mer neo-peptide (SEQ ID NO: 1) was processed and presented to T-cells in the context of HLA-A3 on FGF-5-expressing renal cancers.
An alanine scan was performed on the 9-mer (SEQ 1D NO: 1), using standard methods, to deternzine which amino acids could tolerate an alanine substitution, without significant loss in immunoreactivity. Position 173 of the peptide tolerates alanine substitution, and there is only partial reduction in activity with alanine substitutions at positions 172 (N1~2), 176 (516) and 217 (P21~) (see Table 2). Therefore, epitopes having one or more amino acid substitutions at positions 172 (SEQ ID NO: 4), 173 (SEQ
ID
NO: 3), 176 (SEQ ID NO: 5) or 217 (SEQ ID NO: 6) are' encompassed by this disclosure, as are other sequences with amino acid substitutions.
Table 2: Results of Alanine Scan of SEQ ID NO: 1.
Alanine SubstitutionResultin Se uence SEQ ID NO
9-mer with no substitutionNTYASPRFK SEQ ID NO: 1 (amino acids 172-176 and 217-220 of FGF-5) T173A N_AYASPRFK SEQ ID NO: 3 N172A _ATYASPRFK SEQ ID NO: 4 S 176A NTYA_APRFK SEQ ID NO: 5 P217A NTYASARFK SEQ ID NO: 6 HLA-A3 Epitope is Generated by Post-Translational Splicing This example describes the methods used to demonstrate the mechanism that generates the 9-mer fusion peptide (SEQ ID 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.
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 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.
To demonstrate that the HLA-A3 epitope (SEQ ID NO: 1) is generated by post-translational protein splicing, the processing and recognition of synthetic peptides from FGF-5 (see FIG. 8A) pulsed onto HLA-A3+ or HLA-A3- EBV transformed B (EBV-B) cells was determined as follows. EBV-B cells were co-incubated with 10 ~,M of the peptide for 3 hours at 37°C. After the incubation, EBV-B cells were washed and CTL
were added. IFN-y in the supernatant was measured 20-24 hours later by ELISA.
Peptide pulsing was done in RPMI and the co-culture was done in RPMI with 10%
FBS. Where indicated, EBV-B cells were fixed in 1% formaldehyde in PBS, for 10 minutes on ice, and washed in PBS. Peptides were synthesized and molecular weights verified as described in Example 1.
As shown in FIGS. 8B and 8C, high concentrations of a peptide corresponding to FGF-5 172-220 starting with the 5-mer (172-176, NTYAS; amino acids 1-5 of SEQ
ID NO: 1) and ending with the 4-mer (217-220, PRFK; amino acids 6-9 of SEQ ID
NO:
1) activated Clone 2 (but not another RCC-specific CTL clone) in a HLA-A3 restricted manner. Recognition was lost by deletion of residue 172 or residues 213-220 and was not affected by the addition of residues 161-171.
Presentation of the long peptide (SEQ ID NO: 13) was abrogated by mild aldehyde fixation of APCs, a process that did not affect the presentation of the 9-mer (SEQ ID NO: 1) (FIG. 9). Therefore, the results shown in 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).
To confirm this observation, synthetic or acid-stripped 9-mer (NTYASPRFK;
SEQ ID NO: 1) and 49-mer (172-220; SEQ m NO: 13) peptides were fractionated by HPLC and the capacity of fractions to activate Clone 2 determined as follows.
and COS-A3/FGF-5 were prepared by retrovirally transducing COS-T cells. Each cell line was cultured in T175 flasks and the peptides were stripped by adding 7.5 ml/flask of citrate buffer (0.13M citric acid, 0.056M sodium phosphate dibasic, pH 3.1) for 90 seconds. Peptide solutions (from 4x109 cells, 1,200 ml each) were prepared and were concentrated by Sep-pak Plus Cl8 column (Waters). After lyophilization and resolubilization into 200 ~l of 5% acetonitrile 0.05% (v/v) TFA, peptides were fractionated using a C1g 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) . Fractions (1 ml each) were collected, lyophilized, reconstituted in 100 ~1 of RPMI and added to EBV-B. After 3 hours incubation, CTL
in 100 ~,l of RPMI with 20% FBS were added and IFN-y was measured at 20 hours.
Synthetic peptides (9-mer 20 ~,g and 49-mer 100 ~,g) were HPLC fractionated and assayed in the same way as above. All the 9-mer fractions were diluted 100 times before the assay.
As shown in FIGS. l0A and l OB, the antigenic activities of the synthetic 9-mer (SEQ ID NO: 1) and 49-mer (SEQ ID NO: 13) eluted in fractions 11 and 22, respectively. These fractions contained the expected peptides as determined by mass-spectrometry. The 9-mer eluting in fraction in 11 was active using fresh or fixed APCs, while the 49-mer eluting in fraction 22 was active only using live cells.
HPLC also demonstrated that the 9-mer (SEQ ID NO: 1) represents the naturally processed peptide from FGF-5. As shown in FIG. l OD, antigenic activity of acid-s stripped peptides from COS-A3 cells expressing FGF-5 was exclusively recovered from fraction 11. By contrast, none of the fractions from non-FGF-5 expressing cells demonstrated significant antigenic activity (FIC. 1 OC). These results indicate that the naturally processed 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.
Given the unusual origin of the 9-mer determinant (SEQ ID NO: 1), the antigen processing pathway utilized to generate it from full length FGF-5 was determined. An autologous RCC cell line that expresses melanoma antigen gp100, HLA-A2, A3, and DR(31 * 0401 was generated by retroviral vector-mediated transduction of gp 100, HLA-A2, and class II transactivator CIITA genes (CIITA), (the tumor is genotypically HLA-DR(31 * 0401 ). The result is to create a tumor target capable of presenting not only its autologous antigens to Clone 2, but also class I and II-restricted determinants from gp100 to control T-cell clones which recognize this melanoma antigen (class II
gp100 recognition is restricted by DR[31 *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 p.M clasto-Lactacystin (3-Lactone (Calbiochem) for 3 hours.
After washing, T-cells were added and IFN-y was measured at 20 hours using the methods described in Example 1. TAP-1 dependency was determined by infecting the RCC
line with either an adenovirus encoding GFP or TAP-1 inhibitor ICP47 (MOI=100, 2 hours).
After washing, overnight culture and the treatment with the citric acid buffer as above, T-cells were added, and IFN-y measured at 20 hours. A CD8+ T cell clone that recognizes the gp100209-217 determinant was used as a positive control for inhibitor effectiveness and a CD4+ T cell clone that recognizes gp1OO44-59 determinant was used as a control to eliminate non-specific effects of inhibitors on general antigen presentation capacity.
As shown in FIGS. 11A and 11B, presentation ofboth MHC class I-restricted peptides was blocked by the proteasome inhibitor clasto-Lactacystin [3-lactone or by expressing ICP47 to inhibit TAP-mediated cytosol to ER peptide transport. MHC
class II-restricted gp100 recognition was unaffected.
Based on these results, it appears that the 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. StYUCt. Biol. 1:502-4). The proteasome and TAP-dependence of antigen presentation and the successful splicing of plasmid encoded trunctated 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.
These results also demonstrate that the immune system monitors non-contiguous peptide sequences generated post-translationally. This capability represents an enormous increase in the ability of CTLs to recognize self and foreign proteins. While this could enhance immune surveillance, it does complicate the task of identifying peptide ligands recognized by tumor- and pathogen-specific CTL.
Identification of an HLA-A2-Restricted Epitope To broaden the subject base that could participate in vaccine trials targeting FGF-5, an HLA-A2-restricted epitope of FGF-5 was identified using the following methods.
Peripheral blood lymphocytes (PBL) from HLA-A2 subj ects 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.
Collins, CO), that was reportedly a peptide from the N-terminal leader sequence of FGF-5, and repetitively stimulating PBL from an HLA-A2 positive RCC subject using the synthetic peptide preparation containing FGF-5 sequences, a culture which recognized the stimulating peptide preparation pulsed onto A2+ cells (but not a control peptide) and recognized an FGF-5 expressing HLA-A2+ RCC line (but not the same tumor without HLA-A2) was obtained. This was cloned by limiting dilution and six clones with the same reactivity were obtained. These clones recognized peptide, FGF-5+/HLA-A2+ tumor as well as HLA-A2+ COS cells transfected with the gene for FGF-5, but not the control vector, confirming that the peptide epitope was naturally processed from full length FGF-5 and presented on HLA-A2 (FIG. 12).
Unfortunately, mass spectroscopy of preparation 5194 demonstrated that it was not pure, and instead contained a number of peptides. The actual peptide recognized was determined by testing truncations of the FGF-5 gene transfected into HLA-A2+
cells. A2+ target cells transfected with the full-length FGF-5 gene were recognized.
Truncations of the FGF-5 gene localized the epitope to a region overlapping nucleotides 467-541 of FGF-5 (Genbank Accession No: M37825). A candidate A2-binding 10-rner 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).
To further demonstrate that 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 (L118F) at position two of SEQ ID 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.
Overview of Methods of Using FGF-5 Epitopes Having demonstrated that tumor-reactive T-cells generated from subjects with RCC can recognize naturally presented FGF-5 in either the context of HLA-A2 or HLA-A3 via the minimal determinants MLSVLEIFAV (SEQ ID NO: 7) or NTYASPRFK (SEQ ID NO: 1), respectively, such 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.
' Disclosed in the examples below are methods that one skilled in the art can use to determine response rates and toxicity of peptide vaccination with SEQ ID
NO: 1 or 7 (or variants or fusions thereof) in HLA-appropriate subjects with RCC and determine the effect of such administration on the response rate to high-dose IL-2. This is done as vaccine-only in subjects who are not candidates for IL-2 therapy or with IL-2 therapy in subjects who are eligible to receive 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 (IFA) 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. They receive vaccination and high-dose bolus IL-2 along with peptide (in the presence of IFA) vaccine with clinical response and immunological response to vaccination as primary endpoints.
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.
Administration of FGF-5 Peptides 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. In this example, 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 A begin receiving vaccination with HLA-appropriate peptide emulsified in IFA every 3 weeks. This is continued for up to a year, or until tumor progression (see Example 9) is documented. At that point, those eligible for IL-2 who have not yet received it, can have high-dose intravenous bolus IL-2 added to their peptide vaccination regimen. Throughout, all IL-2 is administered at 720,000 IU/kg/dose every 8 hours by intravenous bolus to the maximum tolerated number of doses (which constitutes one cycle of therapy). Two cycles, separated by 10-14 days, is administered during every two month period, with further treatment dependent on interval tumor assessment. For subjects in Cohort A crossing over to vaccination plus IL-2 therapy, a single peptide + IFA vaccination is administered the day prior to starting an IL-2 cycle (instead of every three weeks, to accommodate the standardized regimen).
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.
Subjects with advanced loco-regional disease have a poor prognosis and no adjuvant therapy options of efficacy. Therefore, Cohort C includes subjects who have undergone resection of either T3/T4 or Nl/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.
Within each cohort, 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 ID
NO: l, 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 (SEQ ID NO: 1) in IFA. The 1 mg dose of peptide was selected based on immunization with peptides derived from the MART-1 and gp100 melanoma antigens, where no significant dose response relationship was observed over 0.1 mg to 10 mg of peptide (Cormier et al., 1997. Cancer J. Sci. Am. 3:37-44; Salgaller et al., 1996 Cancer Res.
56:4749-57).
Table 3: Examples of Treatment Methods (Each cohort has subgroups for HLA-A2 and HLA-A3) Cohort A Cohort B Cohort C
Metastatic disease Metastatic disease - Resected Stage III
disease able to delay IL-2 receives immediate Starts vaccine aloneStarts standard Starts vaccine once IL-2 with alone every 3wks vaccine rior to once every 3wks each cycle If PD seen, and If PD seen, patientIf relapse seen, IL2- taken without eligible, start off of protocol surgical option, standard IL- starts 2 with vaccine prior standard IL-2 to with each cycle vaccine prior to each cycle Evaluations once once every 2 monthsonce every 3 months every 6 while weeks on peptide on IL-2; once everywhile on peptide and once 3-6 once every 3-6 months months if stable every 2 months if stable after while beyond 6 months; , on IL-2; once once every 3-6 every 2 months while months if stable on after Feptides 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 Systems (San Diego, CA). 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; O.1N HCl 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
675756) and sterile water for injection in the anterior thigh deep subcutaneous tissue as follows. To prepare a 1 mg dose, add 1.5 ml of Montanide ISA-51 to a vial of the peptide (1 mg/ml, 1.5 ml ml/vial). The vial is vortexed for 12 minutes, and 2.0 ml is withdrawn for administration.
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.
Other adjuvants can be used, for example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
Ihterleuki~a-2: Iht~avenous Administration In particular examples, IL-2 (Chiron Corp., Emeryville, CA) is administered at a dose of 720,000 ICT/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 rnl 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 xnl of 5% human serum albumin (HSA).
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 mp~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.
Evaluation During Treatment In a particular example, 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 administered as an outpatient, unless inpatient admission is indicated for treatment of vaccine-related side-effects or underlying disease management.
Subjects are monitored after each injection with vital signs twice within the first hour and are discharged from clinic at that time if stable. 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.
Subject peripheral blood lymphocytes (PBL) can be purified by centrifugation on a Ficoll cushion, then incubated in the presence of the vaccinating peptide, and separately in the presence of a control peptide, to stimulate proliferation of specific CTL precursors, as previously described (Cormier et al., 1997. Cancer J. Sci.
Am. 3:37-44; Salgaller et al., 1996 Cancer Res. 56:4749-57). 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.
Evaluation Following Treatment For subjects in cohort A on peptide vaccine alone, complete physical evaluation, CBC, acute care, hepatic and mineral panels and appropriate X-ray evaluations of all evaluable lesions are obtained every six weeks during the first six months of therapy and if stable, every 3-6 months thereafter. Other evaluations can be performed as indicated by symptoms or physical findings.
For cohorts A and B during peptide vaccine plus high-dose IL-2 therapy, complete physical evaluation, CBC, acute care, hepatic and mineral panels and appropriate X-ray evaluations of all evaluable lesions are obtained every two months while on IL-2, and every 3-6 months for stable subjects off therapy.
For cohort C, complete physical evaluation, CBC, acute care, hepatic and mineral panels are performed every three months for the first year and every six months thereafter. Surveillance CT of the chest, abdomen and pelvis are obtained with at least every other assessment and as indicated by symptoms or physical findings.
Additional Treatment In particular examples, if 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.
Evaluation of Responsive Criteria Evaluatioia of target lesiofas All 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 fiu~iher characterize the objective tumor response of the measurable dimension of the disease as outlined in Table 4.
Table 4: Measurement of Target Lesion Response.
Complete Response Disappearance of all target lesions (CR) Partial Response At least a 30% decrease in the sum (PR) of the longest diameter (LD) of target lesions taking as reference the baseline sum LD.
Progression (PD) At least a 20% increase in the sum of LD of target lesions taking as reference the smallest sum LD
recorded since the treatment started or the appearance of one or more new lesions.
Stable Disease Neither sufficient shrinkage to qualify (SD) for PR nor sufficient increase to qualify for PD taking as references the smallest sum LD.
Mixed Res onse A shrinka a of some lesions but an increase in others Evaluation of uou-target lesions All other lesions (or sites of disease) are identified as non-target lesions and recorded at baseline. Measurements are not required, and these lesions are followed as "present" or "absent."
Table 5: Measurement of Non-Target Lesion Response.
Complete Response Disappearance of all non-target (CR): lesions and normalization of tumor marker level.
Non-Com lete Res Persistence of one or more non-target onse: lesions Progression (PD): Appearance of one or more new lesions.
Unequivocal progression of existing non-target lesions Evaluation of best ~verall respofzse The best overall response is the best response recorded from the start of the treatment until disease progressionlrecurrence (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.
Table 6: Evaluation of Overall Response.
Tar et Lesions Non-Tar et LesionsNew Lesions Overall Res onse CR CR No CR
CR Non-CR/Non-PD No PR
PR Non-PD No PR
SD Non-PD No SD
PD Any Yes or No PD
Any PD Yes or No PD
Any An Yes PD
Coufirmatory MeasurerzzeutlDuration of Response Confirmation To be assigned a status of PR or CR, changes in tumor measurements are confirmed by repeat studies that are performed at least 4 weeks after the criteria for response are first met. ~ In the case of SD, follow-up measurements met the SD
criteria at least once after study entry at a minimum interval of 6-8 weeks.
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.
Duration of stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started.
Administration of FGF-5 HLA-A2 and HLA-A3 Epitopes 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+. However, one skilled in the art will understand that other 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. In addition to administering only an FGF-5 HLA-A3 or -A2 epitope to a subject, 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+
For example, using the methods described in Example 5, a combination of FGF-5 HLA-A3 and -A2 epitopes are administered to a subject at a therapeutically effective dose. In a particular example, 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-peptide (such as SEQ ID NO: 1, 9, 17, or a variant, fragment, or fusion thereof) in IFA.
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 -peptides, the FGF-5 expressing or overexpressing tumor can be monitored for regression.
Administering the FGF-5 HLA-AZ and HLA-A3 epitopes of the present disclosure can be accomplished by any means known to the skilled artisan. For example, a pharmaceutically acceptable carrier can be provided for the FGF-5 and HLA-A3 epitopes disclosed herein. However, a pharmaceutically acceptable carrier may not be required to induce an immune response to the FGF-5 HLA-A2 or HLA-A3 epitope. Examples of 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. Thus, a wide variety of pharmaceutically acceptable Garners 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. If 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. In one example, 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. In other words, 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. Alternatively, 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 (C2 -Clo) 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. Usually, 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.
In one example, FGF-5 HLA-A2 or HLA-A3 eptitopic peptides (or combinations thereof) 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 i~ 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, Treyads 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.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
Illustrative phospholipids include egg phosphatidylcholine, dipalinitoylphosphatidylcholine 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.
In the case of a liposomal targeted delivery system, 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.
In one example, a liposome includes the desired peptide and is directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic epitope. A variety of methods are available for preparing liposomes and are described, for example, in Szoka et al., 1980, Ann. Rev. Biophys. Bioeng 9:467 and U.S.
Pat. Nos.
4,235,871, 4,501,728, 4,837,028, and 5,019,369. 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. For example, 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.
For administration of nucleic acid molecules, 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. In one example, the retroviral vector is a derivative of a marine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney marine leukemia virus (MoMuLV), Harvey marine sarcoma virus (HaMuSV), marine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). When the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) can be utilized. A
number of additional 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. By inserting 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. Preferred targeting is accomplished by using 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.
Since recombinant retroviruses are defective, they need assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using 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.
The pharmaceutical 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. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science 249:1527-33, 1990 (incorporated herein by reference).
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. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
Various considerations are described, e.g., in Gilinan et al., eds., Goodman and Gilman:
Tlae Pharmacological Bases of Therapeutics, ~th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17~' ed., Mack Publishing Co., Easton, Pa., 1990. Typically, 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. In one example, the dose is about 1.0 p,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. In one example, 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. In the case of a more aggressive disease 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.
In one example, purified therapeutically active molecules are combined with 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. In general, the nature of the pharmaceutically acceptable carrier will depend on the particular mode of administration being employed. For instance, 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. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, 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.
Pharmaceutical preparations 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.
As is known in the art, protein-based pharmaceuticals may be only inefficiently delivered through ingestion. However, 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 administering 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.
For oral administration, 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%.
For aerosol adnvnistration, 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. Mixed esters, such as mixed or natural glycerides may also be employed.
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.
Peptide Modifications 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. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1RZ wherein Rl and RZ are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, malefic, tartaric and other organic salts, or may be modified to Cl-C16 allcyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the peptide side chains may be converted to C1-Cls alkoxy or to a Cl-C16 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 C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and,esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the disclosed peptides to select and provide conformational constraints to the structure that result in enhanced stability.
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. For computer modeling applications, 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). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press:
Buffalo Grove, IL, pp. 165-174 and Py~inciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CARD. Also included within the scope of the disclosure are mimetics prepared using such techniques. In one example, a mimetic mimics the immune response generated by an FGF-5 HLA-A3 or HLA-A2 epitope.
Determination of a Therapeutically Effective Amount This example describes methods that can be used to identify different therapeutically effective doses of an FGF-5 HLA-A3 epitope, FGF-5 HLA-A2 epitope, or combinations thereof. In one example, 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.
One example of 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.
In another or additional example, it is an amount sufficient to increase the efficacy of another agent, such as IL-2. In one example, 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.
An effective amount of FGF-5 HLA-A3 or HLA-A2 epitope protein, autologous CTLs specific to FGF-5, or combinations thereof, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, 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 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. For example, 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 methods disclosed herein have equal application in medical and veterinary settings. Therefore, 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.
In view of the many possible embodiments to which the principles of our disclosure may be applied, it is be recognized that the illustrated embodiments are only examples of the disclosure and are not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
SEQUENCE LISTING
<110> The Government of the United States of America <120> IMMUNOGENIC EPITOPES FOR FIBROBLAST GROWTH FACTOR 5 (FGF-5) <130> 67015 <150> US 60/427,920 <151> 2002-11-19 <160> 17 <170> PatentIn version 3.2 <210> 1 <211> 9 <212> PRT
<213> homo sapiens <400> 1 Asn Thr Tyr Ala Ser Pro Arg Phe Lys <210> 2 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Artifical peptide sequence.
<400> 2 Asn Thr Tyr Ala Ser Leu Pro Arg Phe Lys <210> 3 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 3 Asn Ala Tyr Ala Ser Pro Arg Phe Lys <210> 4 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 4 Ala Thr Tyr Ala Ser Pro Arg Phe Lys <210> 5 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 5 Asn Thr Tyr Ala Ala Pro Arg Phe Lys <210> 6 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant FGF-5 HLA-A3 epitope.
<400> 6 Asn Thr Tyr Ala Ser Ala Arg Phe Lys <210> 7 <211> 10 <212> PRT
<213> homo Sapiens <400> 7 Met Leu Ser Val Leu Glu Ile Phe Ala Val <210> 8 <211> 10 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 8 Met Phe Ser Val Leu Glu Ile Phe Ala Val <210> 9 <211> 6 <212> PRT
<213> homo Sapiens <400> 9 Tyr Ala Ser Pro Arg Phe <210> 10 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 10 Asn Thr Tyr Phe Leu Pro Arg Phe Lys <210> 11 <211> 48 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 11 Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys <210> 12 <211> 52 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 12 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser <210> 13 <211> 49 <212> PRT
<213> Artificial Sequence <220> .
<223> Variant peptide sequence.
<400> 13 Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe 35 40 45 , Lys <210> 14 <211> 60 <212> PRT
<213> Artificial Sequence <220>
<223> Variant peptide sequence.
<400> 14 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys <210> 15 <211> 1123 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (140)..(943) <400> 15 cctctcccct tctcttcccc gaggctatgt ccacccggtg cggcgaggcg ggcagagcca 60 gaggcacgca gccgcacagg ggctacagag cccagaatca gccctacaag atgcacttag 120 gacccccgcg gctggaaga atg agc ttg tcc ttc ctc ctc ctc ctc ttc ttc 172 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe agc cac ctg atc ctc agc gcc tgg get cac ggg gag aag cgt ctc gcc 220 Ser His Leu Ile Leu Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala ccc aaa ggg caa ccc gga ccc get gcc act gat agg aac cct ata ggc 268 Pro Lys Gly Gln Pro Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly tcc agc agc aga cag agc agc agt agc get atg tct tcc tct tct gcc 316 Ser Ser Ser Arg Gln Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala tcc tcc tcc ccc gca get tct ctg ggc agc caa gga agt ggc ttg gag 364 Ser Ser Ser Pro Ala Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu cag agc agt ttc cag tgg agc ccc tcg ggg cgc cgg acc ggc agc ctc 412 Gln Ser Ser Phe Gln Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu tac tgc aga gtg ggc atc ggt ttc cat ctg cag atc tac ccg gat ggc 460 Tyr Cys Arg Val Gly Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly aaa gtc aat gga tcc cac gaa gcc aat atg tta agt gtt ttg gaa ata 508 Lys Val Asn Gly Ser His Glu Ala Asn Met Leu Ser Val Leu Glu Ile ttt get gtg tct cag ggg att gta gga ata cga gga gtt ttc agc aac 556 Phe Ala Val Ser Gln Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn aaa ttt tta gcg atg tca aaa aaa gga aaa ctc cat gca agt gcc aag 604 Lys Phe Leu Ala Met Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys ttc aca gat gac tgc aag ttc agg gag cgt ttt caa gaa aat agc tat 652 Phe Thr Asp Asp Cys Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr aat acc tat gcc tca gca ata cat aga act gaa aaa aca ggg cgg gag 700 Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu tgg tat gtt gcc ctg aat aaa aga gga aaa gcc aaa cga ggg tgc agc 748 Trp Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser ccc cgg gtt aaa ccc cag cat atc tct acc cat ttt ctt cca aga ttc 796 Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe aag cag tcg gag cag cca gaa ctt tct ttc acg gtt act gtt cct gaa 844 Lys Gln Ser Glu Gln Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu aag aaa aat cca cct agc cct atc aag tca aag att ccc ctt tct gca 892 Lys Lys Asn Pro Pro Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala cct cgg aaa aat acc aac tca gtg aaa tac aga ctc aag ttt cgc ttt 940 Pro Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe gga taatattaat cttggccttg tgagaaacca ttctttcccc tcaggagttt 993 Gly ctataggtgt cttcagagtt ctgaagaaaa attactggac acagcttcag ctatacttac 1053 actgtattga agtcacgtca tttgtttcag tgtgactgaa acaaaatgtt ttttgatagg 1113 aaggaaactg 1123 <210> 16 <211> 268 <212> PRT
<213> Homo Sapiens <400> 16 Met Ser Leu Ser'Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu r Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly <210> 17 <211> 6 <212> PRT
<213> Artificial <220>
<223> Artificial peptide.
<220>
<221> MISC FEATURE
<222> (3) . . (3) <223> Xaa is Ala or Ser <220>
<221> MISC_FEATURE
<222> (4). (4) <223> Xaa is Ala or Pro <400> 17 Tyr Ala Xaa Xaa Arg Phe
Claims
We claim:
1. A purified immunogenic peptide comprising Tyr-Ala-(A3)-(A4)-Arg-Phe wherein A3 is Ala or Ser and A4 is Ala or Pro (SEQ ID NO: 17), wherein the peptide is at least eight amino acids in length.
2. The peptide of claim 1, wherein the peptide comprises anti-FGF-5 expressing or over-expressing tumor activity.
3. The peptide of claim 1, wherein the peptide comprises SEQ ID NO: 9, and wherein SEQ ID NO: 9 comprises one or two amino acid substitutions.
4. The peptide of claim 1, wherein the peptide comprises SEQ ID NO: 1, and wherein SEQ ID NO: 1 comprises one or two amino acid substitutions.
5. The peptide of claim 1, wherein the peptide comprises an amino acid sequence set forth as SEQ ID NO: 1 or 9.
6. The peptide of claim 4, wherein the variant comprises SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
7. The peptide of claim 4, wherein the variant consists of SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
8. The peptide of claim 1, wherein the peptide consists of SEQ ID NO: 1.
9. The peptide of claim 1, wherein the peptide is at least 9 amino acids in length.
10. The peptide of claim 1, wherein the peptide is 8-500 amino acids in length.
12. A purified immunogenic peptide comprising at least 78% sequence identity to SEQ ID NO: 1, wherein the peptide is at least nine amino acids in length.
13. A purified immunogenic peptide comprising a sequence having at least 80%
sequence identity to SEQ ID NO: 7.
14. The peptide of claim 13, wherein the peptide comprises SEQ ID NO: 7 with one or two amino acid substitutions.
15. The peptide of claim 13, wherein the peptide comprises SEQ ID NO: 7.
16. The peptide of claim 13, wherein the peptide consists of SEQ ID NO: 7.
17. The peptide of claim 13, wherein the peptide comprises at least 10 amino acids.
18. The peptide of claim 13, wherein the peptide comprises about 10-100 amino acids.
19. An isolated nucleic acid encoding the peptide of claim 1 or claim 13.
20. The nucleic acid of claim 19, operably linked to a promoter.
21. A vector comprising the nucleic acid of clam 19.
22. A host cell transformed with the vector of claim 21.
23. A pharmaceutical composition, comprising a therapeutically effective amount of the peptide of claim 1 or claim 13, or a a therapeutically effective amount of nucleic acid encoding the peptide of claim 1 or claim 13.
24. A pharmaceutical composition, comprising a therapeutically effective amount of the peptide of claim 1 and the peptide of claim 13.
25. A pharmaceutical composition, comprising a a therapeutically effective amount of a nucleic acid encoding the peptide of claim 1 and a therapeutically effective amount of a nucleic acid encoding the peptide of claim 13.
26. The pharmaceutical composition of claim 23, further comprising an adjuvant.
27. The pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable carrier.
28. A method of eliciting an immune response in a subject, comprising administering to the subject a first dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both.
29. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A2 epitope, and the peptide of claim 13 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A2 epitope.
30. The method of claim 28, wherein the method is a method of eliciting an immune response against a renal cell carcinoma (RCC).
31. The method of claim 29, wherein the peptide administered comprises an amino acid sequence set forth as SEQ ID NO: 7.
32. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A3 epitope and the peptide of claim 1 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A3 epitope.
33. The method of claim 32, wherein the peptide administered comprises SEQ
ID NO: 1; SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
34. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A2 epitope and an FGF-5 HLA-A3 epitope, and the peptide of claim 13 is administered resulting in elicitation of the immune response against the FGF-5 epitope, and the peptide of claim 1 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A3 epitope.
35. The method of claim 28, wherein the subject has an FGF-5 expressing tumor.
36. The method of claim 35, wherein the tumor is an adenocarcinoma.
37. The method of claim 35, wherein the tumor is a renal cell carcinoma (RCC).
38. The method of claim 28, wherein an HLA haplotype of the subject is determined prior to administering to the subject a therapeutically effective amount of the peptide, wherein if the subject has an HLA-A2 haplotype, the subject is administered a therapeutically effective amount of the peptide of claim 13, and wherein if the subject has an HLA-A3 haplotype, the subject is administered a therapeutically effective amount of the peptide of claim 1, 39. The method of claim 28, further comprising determining whether the subject has an FGF-5 expressing tumor.
40. The method of claim 28, further comprising administering a second dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both, at a time after the first dose.
41. A method of treating an FGF-5 expressing tumor in a subject, comprising administering to the subject a therapeutically effective amount of the peptide of claim 1, claim 13, or both, thereby treating the FGF-5 expressing tumor in the subject.
42. The method of claim 41, wherein treatment of the FGF-5 expressing tumor results in a regression of the tumor.
43. The method of claim 41, further comprising administering a therapeutically effective amount of IL-2 to the subject.
44. The method of claim 41, wherein an HLA haplotype of the subject is determined prior to administering to the subject a therapeutically effective amount of the peptide.
45. The method of claim 41, further comprising administering a second dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both at a time after the first dose.
46. A method of generating antibodies specific for an FGF-5 antigen, comprising introducing into a subject the peptide of claim 1 or claim 13.
47. Use of a peptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 to treat an FGF-5 expressing tumor.
48. Use of a peptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 to elicit an immune response.
1. A purified immunogenic peptide comprising Tyr-Ala-(A3)-(A4)-Arg-Phe wherein A3 is Ala or Ser and A4 is Ala or Pro (SEQ ID NO: 17), wherein the peptide is at least eight amino acids in length.
2. The peptide of claim 1, wherein the peptide comprises anti-FGF-5 expressing or over-expressing tumor activity.
3. The peptide of claim 1, wherein the peptide comprises SEQ ID NO: 9, and wherein SEQ ID NO: 9 comprises one or two amino acid substitutions.
4. The peptide of claim 1, wherein the peptide comprises SEQ ID NO: 1, and wherein SEQ ID NO: 1 comprises one or two amino acid substitutions.
5. The peptide of claim 1, wherein the peptide comprises an amino acid sequence set forth as SEQ ID NO: 1 or 9.
6. The peptide of claim 4, wherein the variant comprises SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
7. The peptide of claim 4, wherein the variant consists of SEQ ID NO: 3, SEQ
ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
8. The peptide of claim 1, wherein the peptide consists of SEQ ID NO: 1.
9. The peptide of claim 1, wherein the peptide is at least 9 amino acids in length.
10. The peptide of claim 1, wherein the peptide is 8-500 amino acids in length.
12. A purified immunogenic peptide comprising at least 78% sequence identity to SEQ ID NO: 1, wherein the peptide is at least nine amino acids in length.
13. A purified immunogenic peptide comprising a sequence having at least 80%
sequence identity to SEQ ID NO: 7.
14. The peptide of claim 13, wherein the peptide comprises SEQ ID NO: 7 with one or two amino acid substitutions.
15. The peptide of claim 13, wherein the peptide comprises SEQ ID NO: 7.
16. The peptide of claim 13, wherein the peptide consists of SEQ ID NO: 7.
17. The peptide of claim 13, wherein the peptide comprises at least 10 amino acids.
18. The peptide of claim 13, wherein the peptide comprises about 10-100 amino acids.
19. An isolated nucleic acid encoding the peptide of claim 1 or claim 13.
20. The nucleic acid of claim 19, operably linked to a promoter.
21. A vector comprising the nucleic acid of clam 19.
22. A host cell transformed with the vector of claim 21.
23. A pharmaceutical composition, comprising a therapeutically effective amount of the peptide of claim 1 or claim 13, or a a therapeutically effective amount of nucleic acid encoding the peptide of claim 1 or claim 13.
24. A pharmaceutical composition, comprising a therapeutically effective amount of the peptide of claim 1 and the peptide of claim 13.
25. A pharmaceutical composition, comprising a a therapeutically effective amount of a nucleic acid encoding the peptide of claim 1 and a therapeutically effective amount of a nucleic acid encoding the peptide of claim 13.
26. The pharmaceutical composition of claim 23, further comprising an adjuvant.
27. The pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable carrier.
28. A method of eliciting an immune response in a subject, comprising administering to the subject a first dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both.
29. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A2 epitope, and the peptide of claim 13 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A2 epitope.
30. The method of claim 28, wherein the method is a method of eliciting an immune response against a renal cell carcinoma (RCC).
31. The method of claim 29, wherein the peptide administered comprises an amino acid sequence set forth as SEQ ID NO: 7.
32. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A3 epitope and the peptide of claim 1 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A3 epitope.
33. The method of claim 32, wherein the peptide administered comprises SEQ
ID NO: 1; SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
34. The method of claim 28, wherein the immune response elicited is against an FGF-5 HLA-A2 epitope and an FGF-5 HLA-A3 epitope, and the peptide of claim 13 is administered resulting in elicitation of the immune response against the FGF-5 epitope, and the peptide of claim 1 is administered resulting in elicitation of the immune response against the FGF-5 HLA-A3 epitope.
35. The method of claim 28, wherein the subject has an FGF-5 expressing tumor.
36. The method of claim 35, wherein the tumor is an adenocarcinoma.
37. The method of claim 35, wherein the tumor is a renal cell carcinoma (RCC).
38. The method of claim 28, wherein an HLA haplotype of the subject is determined prior to administering to the subject a therapeutically effective amount of the peptide, wherein if the subject has an HLA-A2 haplotype, the subject is administered a therapeutically effective amount of the peptide of claim 13, and wherein if the subject has an HLA-A3 haplotype, the subject is administered a therapeutically effective amount of the peptide of claim 1, 39. The method of claim 28, further comprising determining whether the subject has an FGF-5 expressing tumor.
40. The method of claim 28, further comprising administering a second dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both, at a time after the first dose.
41. A method of treating an FGF-5 expressing tumor in a subject, comprising administering to the subject a therapeutically effective amount of the peptide of claim 1, claim 13, or both, thereby treating the FGF-5 expressing tumor in the subject.
42. The method of claim 41, wherein treatment of the FGF-5 expressing tumor results in a regression of the tumor.
43. The method of claim 41, further comprising administering a therapeutically effective amount of IL-2 to the subject.
44. The method of claim 41, wherein an HLA haplotype of the subject is determined prior to administering to the subject a therapeutically effective amount of the peptide.
45. The method of claim 41, further comprising administering a second dose of a therapeutically effective amount of the peptide of claim 1, claim 13, or both at a time after the first dose.
46. A method of generating antibodies specific for an FGF-5 antigen, comprising introducing into a subject the peptide of claim 1 or claim 13.
47. Use of a peptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 to treat an FGF-5 expressing tumor.
48. Use of a peptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 to elicit an immune response.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US42792002P | 2002-11-19 | 2002-11-19 | |
US60/427,920 | 2002-11-19 | ||
PCT/US2003/037065 WO2004045555A2 (en) | 2002-11-19 | 2003-11-19 | Immunogenic epitopes for fibroblast growth factor 5 (fgf-5) presented by hla-a3 and hla-a2 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2506684A1 true CA2506684A1 (en) | 2004-06-03 |
Family
ID=32326614
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002506684A Abandoned CA2506684A1 (en) | 2002-11-19 | 2003-11-19 | Immunogenic epitopes for fibroblast growth factor 5 (fgf-5) presented by hla-a3 and hla-a2 |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1562622A2 (en) |
AU (1) | AU2003298677A1 (en) |
CA (1) | CA2506684A1 (en) |
WO (1) | WO2004045555A2 (en) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1225909A2 (en) * | 1999-10-02 | 2002-07-31 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Fibroblast growth factor-5 (fgf-5) is a tumor associated t-cell antigen |
US20020031805A1 (en) * | 1999-12-29 | 2002-03-14 | Conklin Darrell C. | Novel FGF homolog zFGF10 |
-
2003
- 2003-11-19 WO PCT/US2003/037065 patent/WO2004045555A2/en not_active Application Discontinuation
- 2003-11-19 EP EP03796431A patent/EP1562622A2/en not_active Withdrawn
- 2003-11-19 AU AU2003298677A patent/AU2003298677A1/en not_active Abandoned
- 2003-11-19 CA CA002506684A patent/CA2506684A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2004045555A2 (en) | 2004-06-03 |
WO2004045555A3 (en) | 2004-08-26 |
AU2003298677A1 (en) | 2004-06-15 |
EP1562622A2 (en) | 2005-08-17 |
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